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. 2019 May 30;16:298–311. doi: 10.1016/j.isci.2019.05.039

Figure 1.

Figure 1

ICV Injection of Aβ-Th1 Cells to 5XFAD Mice Decreases Aβ Plaques in the Brain Associated with Increased Expression of MHCII by Parenchymal APCs

Aβ-Th1 T cells were generated following the Aβ1–42 immunization of 2-month-old mice (Transparent Methods and Figure S1A). 5XFAD mice were ICV-injected with either Aβ-Th1 T cells or PBS, killed at 11 or 21 days post-injection (dpi), and their brains collected and analyzed by IHC or qPCR.

(A) Illustration of a representative coronal view of the adult mouse brain and the injection site in the lateral ventricle.

(B) A representative IHC image (left) of Aβ plaques co-localized with GFP+ T cells (green) in brain sections derived from 5XFAD mice 11 dpi of activated Aβ-Th1 cells. Sections were immunolabeled with anti-Aβ (red) and a DAPI nucleus counterstain (blue). The middle image is a higher magnification of the framed area, showing the interaction between Aβ and Aβ-Th1 T cells. The right image is a 3D reconstruction of z-sections (9.75 μm overall, 0.75 μm/slice) of the framed area. Scale bars, 200 μm (left), 50 μm (middle), and 5 μm (right).

(C) Representative IHC images showing Aβ plaque load in brain sections derived from 5XFAD mice ICV-injected with either PBS (PBS→AD, left) or Aβ-Th1 T cells (Th1→AD, right). Sections were taken at 21 dpi (n = 4–5 mice per group) and immunolabeled with anti-Aβ plaques (red) and a DAPI nucleus counterstain (blue). Scale bars, 200 μm. The quantitative analysis of the Aβ plaque load in the cortex (right graph) was performed with IMARIS and shows the mean ± SEM results of one experiment out of four performed.

(D) Representative IHC images showing the upregulation of MHCII+ cells in brain sections derived from either PBS→AD (left) or Th1→AD (right) mice at 21 dpi (n = 6 mice per group), immunolabeled with anti-MHCII (green), anti-Aβ (red), and a DAPI nucleus counterstain (blue). The higher magnifications of the framed areas reveal MHCII+ cells accumulating near the Aβ plaques. Scale bars, 200 μm in the low-magnification images and 50 μm in the high-magnification images. The quantitative analysis of the number of MHCII+ cells per volume of cortical section (right graph) was performed with IMARIS. Each symbol represents an individual mouse, whereas the horizontal lines indicate the mean ± SEM of one experiment out of four performed.

(E) qPCR analysis of CD74 expression in the cortex (left) and hippocampus (right) of Th1→AD and PBS→AD mice at 21 dpi (n = 6–7 mice per group). Bars represent means ± SEM.

(F) Representative IHC images demonstrating an immunological synapse between MHCII+ cells and GFP+ T cells in brain sections derived from Th1→AD mice at 11 dpi (n = 5 mice) and immunolabeled with anti-phalloidin (cytoskeletal marker, blue), anti-MHCII (red), and GFP T cells (top images), or with anti-Zap70 (phospho Tyr 319) (red), anti-MHCII (blue), GFP T cells, and a DAPI nucleus counterstain (gray) (bottom images). In the top images, the middle and right panels are 3D reconstructions of z-sections (11.25 μm overall, 0.75 μm/slice). Scale bars, 10 μm. In the bottom images, the middle and right images are a 3D reconstruction of z-sections (7 μm overall, 0.5 μm/slice), which was then sliced in the xy axis. The front orientation of the xy-sliced 3D reconstruction shows an immunological synapse via Zap70 (phospho Tyr 319) (right). Scale bars, 10 μm (left) and 5 μm (middle and right images). *p < 0.05, **p < 0.01, ***p < 0.001 ((C and D) Student's t test and (E) one-way ANOVA).