CD103-CD8+ T cells promote neurotoxic inflammation in Alzheimer's disease via granzyme K-PAR-1 signaling

Abstract

Immune mechanisms contribute to the neuropathology of Alzheimer's disease (AD) but the role of adaptive immune cells is unclear. Here we show that the brain CD8⁺ T cell compartment is dysregulated in AD patients and in the 3xTg-AD mouse model, accumulating activated CD103^- tissue-resident memory T cells that produce large amounts of granzyme K (GrK). These CD103^-CD8⁺ T cells originate from the circulation and migrate into the brain using LFA-1 integrin. Ablation of brain CD103^-CD8⁺ T cells in 3xTg-AD mice ameliorates cognitive decline and reduces neuropathology. GrK induces neuronal dysfunction and tau hyperphosphorylation in human and mouse cells via protease-activated receptor-1 (PAR-1), which is expressed at higher levels in the AD brain, revealing a key immune-mediated neurotoxic axis. We conclude that communication between CD8⁺ T cells and the nervous system is altered in AD, paving the way for therapies targeting T cell-dependent neurotoxic inflammation.

Fig. 1. Characterization of CD8 T cell phenotype in 3xTg-AD and WT mice.

a Graphical overview of the scRNAseq experimental design created in BioRender. Terrabuio, E. (2025) https://BioRender.com/f3lmkb2. b UMAP plot showing CD45^HIGH leukocytes detected in the brains and meninges of wild-type (WT; n = 8) and 3xTg-AD (n = 8) mice. CD8⁺ T cells (n = 3,098) are shown in red. c UMAP plots showing the five subsets of CD8⁺ T cells detected in the brains and meninges of WT (n = 8) and 3xTg-AD (n = 8) mice. d Bubble plot reporting the phenotypic marker genes for each CD8⁺ T cell subset. Transcript levels are color-coded. e Normalized expression of known marker genes on UMAP plot. Transcript levels are color-coded. f Violin plots showing the expression of marker genes for each CD8⁺ T cell subset. White dashed line indicates the median expression level.

Fig. 2. The CD8⁺ Trm compartment is altered in the brains of 3xTg-AD mice.

a, b Violin plots (g) and bubble plot (h) showing the expression of phenotypic markers detected by flow cytometry in the brains and meninges of WT (n = 3 pools of two organs each) and 3xTg-AD (n = 3 pools of two organs each) mice. White dashed line indicates the median expression level in violin plots (g). MFI expressions in the bubble plot (h) are scaled and color-coded. P-values are based on two-way ANOVA - multiple comparisons. Source data are provided as a Source Data file. c Donut plots indicating the distribution of CD8⁺ T cell subsets in the brains of WT (left) and 3xTg-AD (right) mice d Heat map showing the expression of residency genes in the brains and meninges of WT and 3xTg-AD mice. Transcript levels are color-coded. e Violin plots showing gene expression in the brains of WT (n = 8) and 3xTg-AD (n = 8) mice. f Heat map showing the expression in CD45^HIGH leukocytes of genes in brains of WT (n = 8) and 3xTg- AD (n = 8) mice. Transcript levels are color-coded.

Fig. 3. GrK expression is upregulated in brain CD103^–CD8⁺ Trm cells of 3xTg-AD mice.

a Pseudo-time ordered trajectory plot of CD8⁺ T cells in the brains and meninges of WT and 3xTg- AD mice. b Expression of genes in the trajectory plots. Transcript levels are color-coded. c Trajectory plot indicating the distribution of brain CD103^– and CD103⁺CD8⁺ Trm cells in WT and 3xTg-AD mice. d, e Abundances of brain CD103^– (d) and CD103⁺ (e) CD8⁺ Trm cells in WT (n = 4) and 3xTg-AD (n = 4) mice. Data are means ± SD. P-values based on two-tailed Student’s t-test. f Scatter plot showing KEGG pathway enrichment analysis for CD103^– and CD103⁺ CD8⁺ Trm cells in the brains of 3xTg-AD mice (PD = “Parkinson disease”, ALS = “Amyotrophic lateral sclerosis”). g Bubble plot showing BP-GO GSEA analysis for brain CD103^–CD8⁺ Trm cells in 3xTg-AD mice. h Normalized gene expression on UMAP plot of brain and meningeal CD8⁺ T cells. Transcript levels are color-coded. i Violin plots showing gene expression in CD103^– and CD103⁺CD8⁺ Trm cells in the brain and meninges. j Violin plot showing gene expression in CD8⁺ T cells in the brains of WT and 3xTg-AD mice. k Representative flow cytometry plot showing GrK expression in brain CD8⁺ T cells in WT and 3xTg-AD mice. l Bar plots showing the percentage of GrK^–, GrK^MED, and GrK^HIGH CD103^– CD8⁺ Trm cells in the brains of WT (n = 5) and 3xTg-AD (n = 5) mice. Data are means ± SD. P-values based on two-tailed Student’s t-test. m Scatter plot showing MFIs of CD103 and GrK in CD8⁺ T cells in the brains of WT (n = 5) and 3xTg-AD (n = 5) mice. n, o Representative histograms (n) and violin plots (o) showing GrK MFI for brain CD103^– (red) and CD103⁺ (blue) CD8⁺ Trm cells in 3xTg-AD mice. White dashed lines indicate median expressions. P-values based on two-tailed Student’s t-test. p CD8⁺ T cells (squares) in the brains of WT and 3xTg-AD mice detected by immunofluorescence microscopy. Scale bar = 5 µm or 2 µm for zoomed images.

Fig. 4. GrK⁺CD103^–CD8⁺ Trm cells induce neuronal alterations by engaging PAR-1.

a Graphical overview of the experimental design. Created in BioRender. Terrabuio, E. (2025) https://BioRender.com/ir5h5rf. b Immunofluorescence staining showing GrK granules inside CD103⁺ and CD103^– CD8⁺ Trm cells. Scale bar = 3 µm. c Intracellular Ca²⁺ release in neurons co-cultured with CD103⁺ or CD103^– CD8⁺ Trm cells. The last time point is shown in the right panel. Data are means ± SD from four independent experiments. P-values based on two-way ANOVA-multiple comparisons. Ctrl^– = neurons alone. Ctrl⁺ = ionomycin-stimulated neurons (10 µM). Source data are provided as a Source Data file. d Intracellular Ca²⁺ release in neurons treated with purified active GrK or vehicle. The last time point is shown in the right panel. Data are means ± SD from three independent experiments. P-values based on two-way ANOVA-multiple comparisons. Source data are provided as a Source Data file. e Intracellular Ca²⁺ release in neurons co-cultured for 5 h with CD103^–CD8⁺ Trm cells in the presence/absence of the PAR-1 inhibitor SCH79797 (100 nM). The last time point (300 min) is shown in the right panel. Data are means ± SD from four independent experiments. P-values based on two-way ANOVA-multiple comparisons. Ctrl^– = neurons alone. Ctrl⁺ = ionomycin-stimulated neurons (10 µM). Source data are provided as a Source Data file. f, g Intracellular Ca²⁺ release in neurons cultured for 5 h with purified active GrK alone (150 nM) or with SCH79797 (50 nM, or 100 nM). Ctrl^– = neurons alone. The last time point is shown in the right panel. Data are means ± SD from three independent experiments. P-values based on two-way ANOVA-multiple comparisons. Representative images are shown in (g). Red = intracellular Ca²⁺ release. Scale bar = 20 µm. Source data are provided as a Source Data file. h Immunofluorescence microscopy showing GrK⁺CD103^–CD8⁺ Trm cells near the soma (cell I) and dendrites (cell II) of a PAR-1⁺ neuron. Cell morphology was visualized by wide-field imaging using a DIC filter. Scale bar = 5 µm (or 2 µm for zoomed images I, and II).

Fig. 5. Detrimental CD103^–CD8⁺ Trm cells in the brain originate from the circulation and their depletion ameliorates disease in 3xTg-AD mice.

a Flow cytometry showing the percentage of brain CD103^– and CD103⁺CD8⁺ Trm cells after anti-CD8 antibody treatment. Anti-CD8 (WT, n = 6; 3xTg-AD, n = 9), isotype-control (WT, n = 6; 3xTg-AD, n = 9). Data are means ± SD from two independent experiments and P-values are based on one-way ANOVA-multiple comparisons was used. b Time spent during training (MWM test). Anti-CD8 (WT, n = 10; 3xTg-AD, n = 21), isotype-control (WT, n = 22; 3xTg-AD, n = 18). Data are means ± SD from two-independent experiments (two-way ANOVA-multiple comparisons). c Representative tracking of three mice/group (MWM test). d, e Violin plots showing path efficiency (d) and number of body rotations (e) (MWM test). Data are from two-independent experiments one-way ANOVA-multiple comparisons). f Bar plot showing the percentage of freezing during the CFC test after anti-CD8 treatment. Anti-CD8 (WT, n = 9; 3xTg-AD, n = 16), isotype-control (WT, n = 21; 3xTg-AD, n = 13). Data are means ± SD from two-independent experiments. One-way ANOVA-multiple comparisons was used. g–i Immunohistochemical staining of the hippocampus after CD8 T cell depletion showing Aβ-load (g), the levels of hyperphosphorylated (h) and total (i) tau protein. Anti-CD8 (n = 3), isotype-control (n = 3). Scale bar = 20 µm. Data are means ± SEM. Two-tailed Student’s t-test was used. ROI = 624.7 µm×501.22 µm. j–l ELISA showing Aβ 1−40 and Aβ 1−42 (j) levels of tau hyperphosphorylation (k) and total tau (l) in soluble and insoluble fractions of brain homogenates after anti-CD8 treatment. Anti-CD8 (n = 4), isotype control (n = 4). Data from three-independent experiments are means ± SEM (two-tailed Student’s t-test). m Dot blot showing insoluble oligomeric and fibrillar forms of Aβ in brain homogenates after anti-CD8 treatment. Anti-CD8 (n = 4), isotype-control (n = 4). Data from three-independent experiments are shown as means ± SEM. P-values based on two-tailed Student’s t-test. n Immunofluorescence staining showing hippocampal oligomeric and fibrillar Aβ. Scale bar = 10 µm.

Fig. 6. LFA-1 integrin controls the accumulation of CD8⁺CD103^– cells in the brains of 3xTg- AD mice.

a Flow cytometry showing the percentage of LFA-1⁺ CD8⁺ T cells (left) and corresponding MFIs (right) in the blood of WT (n = 6) and 3xTg-AD (n = 6) mice. Data are means ± SD, with P-values based on two-tailed Student’s t-test. b Flow cytometry showing the percentage of CD45+ leukocytes among live cells. Data are means ± SEM, with P-values based on two-tailed Student’s t-test. c Flow cytometry showing the percentage of CD103^– (left) and CD103⁺ (right) CD8⁺ Trm cells in the brains of 3xTg-AD (n = 6) and 3xTg-AD/Itgal^–/– (n = 6) mice. Data are means ± SEM, with P-values based on two-tailed Student’s t-test. d–f Representative immunohistochemical staining of the hippocampus in 3xTg-AD (n = 3) and 3xTg-AD/Itgal^–/– (n = 3) mice, showing the Aβ load (c), and the levels of hyperphosphorylated (d) and total (e) tau protein. Scale bar = 20 µm. Data are means ± SEM, with P-values based on two-tailed Student’s t-test. g UMAP plot showing immune cell populations in human blood (n = 3 AD patients and n = 2 negative controls, NCs) by scRNAseq. h Normalized expression of CD3G, CD3D and CD3E on a UMAP plot. Transcript levels are color- coded i CD3⁺ T cell cluster subsets: UMAP plot showing CD8⁺ and CD4⁺ T cell subpopulations in human blood (n = 3 AD patients and n = 2 NCs). j Normalized expression of CD8A, CD8B and CD4 on a UMAP plot. Transcript levels are color- coded. k CD8⁺ T cell cluster subsets: UMAP plot showing early active, Tem, and naïve-like CD8⁺ T cells in human blood (n = 3 AD patients and n = 2 NCs). l Radar plot showing AUCell score using known genes. m Heat map reporting the expression values (mean calculated using the Hurdle model) of ITGAL in AD patients (n = 3) and NCs (n = 2). Transcript levels are color-coded.

Fig. 7. GrK⁺CD103^–CD8⁺ Trm cells accumulate in the CSF of AD patients.

a UMAP plot showing leukocyte populations detected in human CSF (n = 4 AD, n = 5 MCI, and n = 9 healthy controls, HCs) by scRNAseq. b Normalized expression of CD3E, CD8B and CD4 in the UMAP plot. Transcript levels are color-coded. c CD8⁺ T cell cluster subsets: UMAP plot showing KLF2⁺ and Trm cell subpopulations in human CSF (n = 4 AD, n = 5 MCI, and n = 9 HCs). d Normalized expression of KLF2, CD69 and PRDM1 in a UMAP plot. Transcript levels are color-coded. e Trm cell cluster subsets: UMAP plot showing CD103^– and CD103⁺ Trm cells in human CSF (n = 4 AD, n = 5 MCI, and n = 9 HCs). f Percentage of CD103^– and CD103⁺ CD8⁺ cells in human CSF (n = 4 AD, n = 5 MCI, and n = 9 HCs). Data are means ± SD. Source data are provided as a Source Data file. g Violin plot reporting GZMK expression in Trm cells of n = 4 AD patients, n = 5 MCI subjects, and n = 9 HCs. The white dot represents median expression. h Normalized expression of GZMK in a UMAP plot of all Trm cells (panel 1) and Trm cells from HCs (panel 2), MCI subjects (panel 3), and AD patients (panel 4).

Fig. 8. GrK⁺CD103^–CD8⁺ Trm cells accumulate in the brains of AD patients.

a Immunofluorescence staining of control (CTRL) and AD human brain tissues. Scale bar = 40 µm, or 15 µm in the zoomed images. b Bar plots showing the percentage of intraparenchymal CD103^–, CD103⁺, and GrK⁺CD103^– CD8⁺ T cells in the hippocampus of HCs (n = 3) and AD patients (n = 3). Data are means ± SD (two-tailed Student’s t-test). Source data are provided as a Source Data file. c Bar plots showing the percentage of intravascular CD103^–and GrK⁺CD103^– CD8⁺ T cells in the hippocampus of HCs (n = 3) and AD patients (n = 3). Data are means ± SD (two-tailed Student’s t-test). Source data are provided as a Source Data file.

Fig. 9. GrK induces functional alterations in human neuronal cells via PAR-1.

a Immunofluorescence staining for βIII-tubulin, MAP-2, pNF-H, Nestin and PAR-1 in undifferentiated and differentiated SH-SY5Y human neuroblastoma cells. b Intracellular Ca²⁺ release in differentiated SH-SY5Y cells cultured in the presence of 150 nM GrK, or 150 nM GrK and 100 nM SCH79797. The last time point is shown in the bottom panel. Ctrl^– = neurons alone. Ctrl⁺ = ionomycin-stimulated neurons (10 µM). Data are means ± SEM from three independent experiments. P-values based on two-way ANOVA-multiple comparisons. c Representative images of differentiated SH-SY5Y cells at different time points cultured without GrK (Ctrl^–), in the presence of 150 nM active GrK, or in the presence of 150 nM active GrK plus 100 nM SCH79797. The red signal shows intracellular Ca²⁺ release. Scale bar = 20 µm.

Fig. 10. GrK induces tau hyperphosphorylation and alteration of signaling pathways in human neuronal cells via PAR-1.

a–c Bar plots and representative images showing tau phosphorylation levels on serine and threonine (AT8, AT100) (d, e) and only threonine (AT180) (f) residues in differentiated SH-SY5Y cells cultured alone (Ctrl^–), in the presence of 100 nM SCH79797, in the presence of 150 nM active GrK alone, and the presence of both. Data are means ± SD of three independent experiments (two-way ANOVA-multiple comparisons). Scale bar = 5 µm. d ELISA showing the levels of phosphorylation on the pS199, pS396, and pT231 residues of tau protein in differentiated SH-SY5Y cells cultured alone (Ctrl^–), in the presence of 100 nM SCH79797, in the presence of 150 nM active GrK alone, and in the presence of both. Data are means ± SD of three independent experiments. P-values are based on two-way ANOVA multiple comparisons. e, f Protein enrichment analysis from three independent proteomic experiments showing the best enriched clusters of terms (h) and terms belonging to the “Alzheimer’s/neurodegeneration” and “kinase” pathways (i) in SH-SY5Y human neuroblastoma cells cultured in the absence of GrK and SCH79797 (Ctrl^–), in the presence of 100 nM SCH79797 alone, in the presence of 150 nM active GrK alone, and in the presence of both. Source data are provided as a Source Data file.