CD8+ T cells exacerbate AD-like symptoms in mouse model of amyloidosis

Abstract

Alzheimer's disease (AD) is linked to toxic Aβ plaques in the brain and activation of innate responses. Recent findings however suggest that the disease may also depend on the adaptive immunity, as B cells exacerbate and CD8⁺ T cells limit AD-like pathology in mouse models of amyloidosis. Here, by artificially blocking or augmenting CD8⁺ T cells in the brain of 5xFAD mice, we provide evidence that AD-like pathology is promoted by pathogenic, proinflammatory cytokines and exhaustion markers expressing CXCR6⁺ CD39⁺CD73^+/- CD8⁺ T_RM-like cells. The CD8⁺ T cells appear to act by targeting disease associated microglia (DAM), as we find them in tight complexes with microglia around Aβ plaques in the brain of mice and humans with AD. We also report that these CD8⁺ T cells are induced by B cells in the periphery, further underscoring the pathogenic importance of the adaptive immunity in AD. We propose that CD8⁺ T cells and B cells should be considered as therapeutic targets for control of AD, as their ablation at the onset of AD is sufficient to decrease CD8⁺ T cells in the brain and block the amyloidosis-linked neurodegeneration.

Keywords: Alzheimer’s disease; Amyloidosis; Aβ response; B cells; CD8(+) T cells.

Figure 1.. CD8⁺ T cells increased in the brain of mice and humans.

(A) FACS staining results for numbers of CD8⁺ and CD4⁺ T cells, (B) numbers (#) and frequency (%) of CD11b⁺ and CD103⁺ CD8⁺ T cells, and (C) numbers of GrB⁺, TNFα⁺, IFNγ⁺ and IL10⁺ CD8⁺ cells in the brain of 8 months (8M) and 12 months (12M) old WT and 5xFAD female mice. (D) Representative immunofluorescence (IF) staining images and (E) quantification of Aβ plaques (6E10) and CD8⁺ T cells in the hippocampus and subiculum of female WT and 5xFAD mice. White quadrants in hippocampus sections are for magnified images on the right (D). Scale bars are for 500 μm, whole hippocampus sections, and 20 μm, magnified images for the selected areas. (F) Representative IF images for Iba1/6E10/CD8 staining (left, scale bar is 20 μm). CD8⁺ T cells are tightly attached to Iba1⁺ microglia in AD, but not WT, hippocampus (3D reconstruction; scale bar is 5 μm, magnified panels in middle). Right panel is for quantification of CD8⁺ T cells-Iba1⁺ microglia complexes (numbers, F). (G and H) Representative IF staining of postmortem human brain tissues from non-AD, MCI and AD people showing the parenchymal presence of CD8⁺ T cells (G, scale bar is 50 μm) and their complexes with Iba1⁺ microglia (H, scale bar is 20 μm). Vasculature is stained with anti-collagen IV antibody (Col IV). Right panels are for quantification of CD8⁺ T cells (G) and their complexes with Iba1⁺ microglia (H). (I) UMAP plots depict the presence of CD4⁺, CD8A⁺ and CD8B⁺ T cells in SEA-AD datasets. (J) Normalization of CD8⁺ T cells in AD individuals in SEA-AD cohort. Each dot in A-C and E-H is for independent sample. n = 4–7 mice per group in A-C; n = 3–7 mice per group in E; n = 3–6 mice per group in F; n = 6 brains per group in G; n = 3 brains per group in H. * P < 0.05, ** P < 0.01, *** P < 0.001; Two-way ANOVA tests were performed for A-C, unpaired t-tests were performed for E, F and J, one-way ANOVA tests were performed for G and H. Holm-Šídák post hoc tests were used for multiple comparisons, and p-values were shown as noted.

Figure 2.. Depletion of CD8⁺ T cells reverses AD-like pathology in 5xFAD mice.

(A) Correlations between CD8⁺ T cell and numbers of Aβ plaque (i), or Iba1⁺ microglia (ii) in the hippocampus after IF staining; and FACS quantified brain CD8⁺ T cell counts and behavioral impairment in cross maze (iii) of WT and 5xFAD mice (10–13 months old female mice). (B-I) Shown are schema (B) and results of the CD8⁺ T cells depletion experiment, including brain CD8⁺ T cell subsets (numbers quantified with FACS, B and C); and open field test (D) and continuous spontaneous alternation rate in cross maze (E). WT and 5xFAD mice (8–10 months old females) were treated with anti-CD8 (αCD8) and control antibody (IgG). In D, left panel is quantification of time spent in the center area of open field (normalized to WT group); right panel is for representative heatmaps for the time spent in open field (center area shown as red circle). (F-I) Shown are representative IF staining images and quantification of Aβ plaques and Iba1⁺ microglia in the hippocampus (F and G) and neurofilament heavy chain (NFH, normalized to 5xFAD/IgG group) in subiculum (H, I). Scale bar is 1mm (F) and 200 μm (upper panel, H) and 50 μm (lower magnified images, H). Arrows and arrowheads in H indicate long neurofilaments and NFH aggregates, respectively. Dots are for independent mice in A-E, G and I, n = 13 mice in A; n = 5–8 mice per group in B-C; n = 10–16 mice per group in D; n = 6–8 mice per group in E; n = 4–12 mice per group in G; n = 8–12 mice per group in I. D, G and I are from 2 independent experiments. Linear regressions were computed for A, one-way ANOVA tests were performed for B-E, G and I. Holm-Šídák post hoc tests were used for multiple comparisons, and p-values were shown as noted.

Figure 3.. Aβ-vaccine increases brain CD8⁺ T cells to exacerbate AD.

Female 2–3 months (A-F) and 7–8 months (G-J) old 5xFAD mice were i.m. immunized with Aβ-CoreS/LNP vaccine and control LNP. Shown are representative IF image of hippocampus stained with anti-CD8 and 6E10 Abs (A, scale bar is 500 μm in whole hippocampus image and 20 μm in magnified images, white square) and numbers of CD8⁺ T cells in hippocampus, dentate gyrus, and subiculum (IF staining, B), activated/exhausted CD8⁺ T cells in the brain (FACS results, C-E), and NFH aggregates in the subiculum (IF staining, F). Representative IF staining images of the subiculum of WT and 5xFAD mice immunized with Aβ-CoreS or control LNP are shown in G (left panels, scale bar 100 μm, and its magnified area, right panels, scale 20 μm) and respective quantification of Aβ plaques (H) and CD8⁺ T cells in the subiculum (I). Activated brain CD8⁺ T cells were quantified with FACS (J). Dots in B-F and H-J are for independent mice. n = 3–4 mice per group for hippocampus quantification and n = 8–9 brain slices from 3 mice per group for dentate gyrus and subiculum quantification in B, C and F ; n = 11–12 mouse brains per group in D and E; n = 3–4 mice per group in H-I; n = 8–11 mice per group in J. One-way ANOVA tests were performed for B-F, H-J. Holm-Šídák post hoc tests were used for multiple comparisons, and p-values were shown as noted.

Figure 4.. B cells induce brain-infiltrating CD8⁺ T cells in 5xFAD mice.

In 5xFAD-BKO mice, the increase of activated CD8⁺ T cells (A) and their activated subsets (B) in the brain of female and male 5xFAD is reversed to the levels of WT mice. Shown are numbers of cells quantified by FACS. tSNE plot showing enrichment of PD1, LAG3, TIM3, CD11b, CD103, CD44, CD73 and CD39-expressing T cells in the brain from 5xFAD mice as compared to 5xFAD-BKO and WT mice as detected by FACS (Red circle, C). FACS dot plots (D) and quantification (E) of CD39⁺CD73⁺ CD8⁺ T cells in the brain of female WT, 5xFAD, and 5xFAD-BKO mice. (F) The percentage of CXCR6 alone or with CD39⁺CD73⁺ and PD1⁺ or Tox1⁺ in CD8⁺ T cells in the brain and cLN of 5xFAD mice with CXCL16 depleted or BKO. (G) CD39⁺CD73⁺ CD8⁺ T cells in the brain and cLN of 5xFAD mice immunized with Aβ-CoreS as in Fig.3G. n = 5–7 female mice and 4–5 male mice per group in A; n = 3–5 mice per group in B and E; n = 5–8 mice per group in F; n = 11–12 mice per group in G; One-way ANOVA tests were performed for B-F, H-J. Holm-Šídák post hoc tests were used for multiple comparisons, and p-values were shown as noted.