Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer's disease

doi:10.1038/s41586-019-1895-7

. 2020 Jan;577(7790):399-404.

doi: 10.1038/s41586-019-1895-7. Epub 2020 Jan 8.

Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer's disease

David Gate^{1

2}, Naresha Saligrama³, Olivia Leventhal⁴, Andrew C Yang^{5

6}, Michael S Unger^{7

8}, Jinte Middeldorp^{4

9

10}, Kelly Chen⁴, Benoit Lehallier^{4

9}, Divya Channappa⁴, Mark B De Los Santos⁴, Alisha McBride^{4

9}, John Pluvinage^{4

11

12}, Fanny Elahi¹³, Grace Kyin-Ye Tam^{4

14}, Yongha Kim^{4

14}, Michael Greicius^{4

14}, Anthony D Wagner^{15

16}, Ludwig Aigner^{7

8}, Douglas R Galasko¹⁷, Mark M Davis^{3

18

19}, Tony Wyss-Coray^{20

21

22

23

24}

Affiliations

¹ Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA. dgate@stanford.edu.
² Veterans Administration Palo Alto Healthcare System, Palo Alto, CA, USA. dgate@stanford.edu.
³ Department of Microbiology and Immunology, School of Medicine, Stanford University, Stanford, CA, USA.
⁴ Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA.
⁵ Department of Bioengineering, Stanford University, Stanford, CA, USA.
⁶ Chemistry, Engineering and Medicine for Human Health, Stanford University, Stanford, CA, USA.
⁷ Institute of Molecular Regenerative Medicine, Paracelsus Medical University, Salzburg, Austria.
⁸ Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria.
⁹ Veterans Administration Palo Alto Healthcare System, Palo Alto, CA, USA.
¹⁰ Department of Translational Neuroscience, University Medical Center Utrecht Brain Center, Utrecht University, Utrecht, The Netherlands.
¹¹ Medical Scientist Training Program, Stanford University School of Medicine, Stanford, CA, USA.
¹² Stem Cell Biology and Regenerative Medicine Graduate Program, Stanford University School of Medicine, Stanford, CA, USA.
¹³ Department of Neurology, Memory and Aging Center, University of California at San Francisco, San Francisco, CA, USA.
¹⁴ Functional Imaging in Neuropsychiatric Disorders Laboratory, Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA.
¹⁵ Department of Psychology, Stanford University, Stanford, CA, USA.
¹⁶ Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA.
¹⁷ Department of Neurosciences, University of California at San Diego, La Jolla, CA, USA.
¹⁸ Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA, USA.
¹⁹ Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA.
²⁰ Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA. twc@stanford.edu.
²¹ Veterans Administration Palo Alto Healthcare System, Palo Alto, CA, USA. twc@stanford.edu.
²² Chemistry, Engineering and Medicine for Human Health, Stanford University, Stanford, CA, USA. twc@stanford.edu.
²³ Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA. twc@stanford.edu.
²⁴ Paul F. Glenn Center for the Biology of Aging, Stanford University School of Medicine, Stanford, CA, USA. twc@stanford.edu.

PMID: 31915375
PMCID: PMC7445078
DOI: 10.1038/s41586-019-1895-7

Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer's disease

David Gate et al. Nature. 2020 Jan.

. 2020 Jan;577(7790):399-404.

doi: 10.1038/s41586-019-1895-7. Epub 2020 Jan 8.

Authors

Affiliations

¹ Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA. dgate@stanford.edu.
² Veterans Administration Palo Alto Healthcare System, Palo Alto, CA, USA. dgate@stanford.edu.
³ Department of Microbiology and Immunology, School of Medicine, Stanford University, Stanford, CA, USA.
⁴ Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA.
⁵ Department of Bioengineering, Stanford University, Stanford, CA, USA.
⁶ Chemistry, Engineering and Medicine for Human Health, Stanford University, Stanford, CA, USA.
⁷ Institute of Molecular Regenerative Medicine, Paracelsus Medical University, Salzburg, Austria.
⁸ Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University, Salzburg, Austria.
⁹ Veterans Administration Palo Alto Healthcare System, Palo Alto, CA, USA.
¹⁰ Department of Translational Neuroscience, University Medical Center Utrecht Brain Center, Utrecht University, Utrecht, The Netherlands.
¹¹ Medical Scientist Training Program, Stanford University School of Medicine, Stanford, CA, USA.
¹² Stem Cell Biology and Regenerative Medicine Graduate Program, Stanford University School of Medicine, Stanford, CA, USA.
¹³ Department of Neurology, Memory and Aging Center, University of California at San Francisco, San Francisco, CA, USA.
¹⁴ Functional Imaging in Neuropsychiatric Disorders Laboratory, Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA.
¹⁵ Department of Psychology, Stanford University, Stanford, CA, USA.
¹⁶ Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA.
¹⁷ Department of Neurosciences, University of California at San Diego, La Jolla, CA, USA.
¹⁸ Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA, USA.
¹⁹ Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA.
²⁰ Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA. twc@stanford.edu.
²¹ Veterans Administration Palo Alto Healthcare System, Palo Alto, CA, USA. twc@stanford.edu.
²² Chemistry, Engineering and Medicine for Human Health, Stanford University, Stanford, CA, USA. twc@stanford.edu.
²³ Wu Tsai Neurosciences Institute, Stanford University, Stanford, CA, USA. twc@stanford.edu.
²⁴ Paul F. Glenn Center for the Biology of Aging, Stanford University School of Medicine, Stanford, CA, USA. twc@stanford.edu.

PMID: 31915375
PMCID: PMC7445078
DOI: 10.1038/s41586-019-1895-7

Abstract

Alzheimer's disease is an incurable neurodegenerative disorder in which neuroinflammation has a critical function¹. However, little is known about the contribution of the adaptive immune response in Alzheimer's disease². Here, using integrated analyses of multiple cohorts, we identify peripheral and central adaptive immune changes in Alzheimer's disease. First, we performed mass cytometry of peripheral blood mononuclear cells and discovered an immune signature of Alzheimer's disease that consists of increased numbers of CD8⁺ T effector memory CD45RA⁺ (T_EMRA) cells. In a second cohort, we found that CD8⁺ T_EMRA cells were negatively associated with cognition. Furthermore, single-cell RNA sequencing revealed that T cell receptor (TCR) signalling was enhanced in these cells. Notably, by using several strategies of single-cell TCR sequencing in a third cohort, we discovered clonally expanded CD8⁺ T_EMRA cells in the cerebrospinal fluid of patients with Alzheimer's disease. Finally, we used machine learning, cloning and peptide screens to demonstrate the specificity of clonally expanded TCRs in the cerebrospinal fluid of patients with Alzheimer's disease to two separate Epstein-Barr virus antigens. These results reveal an adaptive immune response in the blood and cerebrospinal fluid in Alzheimer's disease and provide evidence of clonal, antigen-experienced T cells patrolling the intrathecal space of brains affected by age-related neurodegeneration.

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Conflict of interest statement

Competing interests

D.G., N.S., M.M.D. and T.W.-C. are co-inventors on a patent application related to this work. Patent STDU2–36496/US-1/PRO is for compositions and methods for measuring T cell markers associated with Alzheimer’s disease.

Figures

**Extended Data Fig. 1 |. Clinical measures of subjects from mass cytometry experiments.**
a, Cohort 1 groups were age-matched and included 57 healthy subjects and 23 patients with MCI or AD. b, Cognitive scoring shows significantly decreased cognitive scores in patients with MCI or AD. Note that not all patients with MCI or AD could complete a cognitive exam and these patients are thus not included in this analysis. Unpaired two-sided f-test with Welch’s correction (n = 51 healthy; n = 12 MCI or AD); mean ± s.e.m. c, d, Quantification of CSF AP as a ratio to phosphorylated tau (c) or total tau (d) reveals significantly reduced ratios in patients with MCI or AD. Unpaired two- sided f-test with Welch’s correction (n = 18 healthy, n = 7 MCI or AD); mean ± s.e.m. e, Top, representative MRI images show reduction in cortical grey matter in patients with AD. Middle, bottom, representative MRI images of several brain regions as measured by subcortical segmentation and hippocampal segmentation. Right, quantification of MRI images of patients with MCI or AD compared to control individuals shows significant reductions in the percentage of the intracranial volume (ICV) of brain regions classically associated with AD pathology. CA1, cornu ammonis area 1. Unpaired two-sided t-test; mean ± s.e.m.

**Extended Data Fig. 2 |. Gating strategy for identifying immune cell populations from mass cytometry data.**
A gating strategy was used to identify populations of immune cells by mass cytometry.

**Extended Data Fig. 3 |. Mass cytometry SPADE and CITRUS clustering shows increased numbers of CD8⁺ T_EMRA cells in MCI or AD.**
a, Normalization of mass cytometry data was performed before all analyses. A gating strategy for input into downstream analyses is shown. Live, single leukocytes were selected for analysis. b, Plotting of clusters by P value and fold change of each cluster reveals cluster 63 as the most highly increased cluster among patients with MCI or AD. Clusters are sized according to their percentage of total PBMCs. Unpaired two-sided t-test (n = 57 healthy; n = 23 MCI or AD). c, CITRUS clustering showing significant differentiating populations (top left). Cluster 229992 and its significant daughter populations are outlined. Expression of CD3, CD8 and CD45RA shows that cluster 229992 corresponds to CD8⁺ T_EMRA cells. d, Quantification of cluster 229992 cells as a percentage of total PBMCs for individual subjects. Percentages of this cluster are significantly higher in patients with MCI or AD than healthy control individuals. Unpaired two-sided t-test with Welch’s correction; mean ± s.e.m. e, Marker expression of cluster 229992 shows it to be a CD3⁺CD8⁺CD45RA⁺CD27⁻ T_EMRA population. f, The regularized supervised learning algorithm from CITRUS predicts disease group with a 20% error rate (80% positive predictability). The number of model features increases from left to right. The most predictive model is shown as the lowest cross validation error rate (red line) constrained by the false discovery rate (yellow triangle).

**Extended Data Fig. 4 |. Altered CD8⁺ T cell subsets in PBMCs from patients with MCI or AD.**
a, Mass cytometry plots of live PBMCs show increased abundance of CD3⁺CD8⁺ T cells in patients with MCI or AD. b, Gating of CD3⁺ T cells into CD4 and CD8 populations shows increased prevalence of CD8⁺ T cells in patients with MCI or AD. c, d, Significantly increased effector CD8⁺ T cells (c) and significantly reduced memory CD8⁺ T cells (d) in patients with MCI or AD. Multivariate analysis of covariance (MANCOVA) using age as a covariate, followed by post-hoc pairwise comparisons of estimated marginal means by unpaired two-sided t-test. Bonferroni correction for multiple comparisons. Confidence intervals (CI) of 95% are shown.

**Extended Data Fig. 5 |. Correlations of memory T cell populations with cognitive scores.**
a, Gating strategy for measuring memory T cell populations. b, Linear regression analysis correlating CD8⁺ T cell populations with cognitive scores indicates a positive relationship between T_EM and T_CM CD8⁺ T cells and no relationship with naive cells. The significance of the difference between datasets was measured by ANCOVA. c, The relationship between the percentage of CD8⁺ T_EMRA cells and cognitive score was not influenced by age. The significance of the difference between the two datasets was measured by analysis of covariance (ANCOVA). Pearson’s correlation r values are shown for each group (b, c). d, Gating strategy for T cell stimulation experiments. e, Increased intracellular TNF cytokine response in PMA-ionomycin-stimulated CD8⁺ T cells from patients with MCI or AD. Unpaired two-sided t-test with Welch’s correction (n = 10 healthy; n = 14 MCI or AD); mean ± s.e.m.

**Extended Data Fig. 6 |. Analysis of peripheral CD8+ TEMRA cells by scRNA-seq.**
a, Gating strategy for sorting peripheral CD8⁺ T_EMRA cells for scRNA-seq. b, Differential expression analysis by scRNA-seq of CD8⁺ T_EMRA cells from patients with MCI or AD versus CD8+ TEMRA cells from healthy individuals shows significantly increased expression of genes that are involved in T cell signalling, including *IFITM3, NFKB/A* and *CD8B2*. Violin plots show average log-normalized counts for significantly upregulated genes in MCI or AD CD8⁺ T_EMRA cells by individual patient (n = 7 healthy; n = 6 MCI or AD). c, Violin plots show average log-normalized counts for significantly upregulated genes in MCI or AD CD8⁺ T_emra cells by group. *GAPDH* is shown as a control. Each dot represents a single cell.

**Extended Data Fig. 7 |. Histological analysis of CD8⁺ T cells in hippocampi from patients with AD and from APP/PS1 mice.**
a, A blood vessel in the brain of a control (non-neurological disease) patient shows a lack of extravascular CD8⁺ T cells. Asterisk indicates the blood vessel lumen. b, A CD8⁺ T cell within an Aβ plaque. Scale bar, 5 μm. c, CD8⁺ T cells in the AD-affected hippocampus in close proximity to Aβ plaques. White lines measure the distances from each cell to the nearest plaque centre. Data in a, b were replicated in at least three independent experiments. d, Quantification of the average distance from CD8⁺ T cells to the nearest Aβ plaque. The dashed red line indicates the average of all cells. e, Representative images of the dentate gyrus that were used to quantify CD3⁺CD8⁺ T cells in the hippocampi of control individuals and patients with AD. Inset shows two CD3⁺CD8⁺ T cells. Sizes of the area plots used for quantification are shown for each image. Scale bar, 500 μm. f, Association of a CD8⁺ T cell with NEFH⁺ neuronal processes by immunohistochemistry and 3D modelling in the APP/PS1 mouse model of cerebral amyloidosis. g, Electron microscopy showing an association of a CD8⁺ T cell with neuronal processes. Red arrowheads indicate areas in which the CD8⁺ T cell associates with neuronal processes. Data in e-g were replicated in at least two independent experiments.

**Extended Data Fig. 8 |. Clonal expansion of T cells in the CSF.**
a, Gating strategy for enumerating T cell subtypes in CSF from healthy elderly individuals. b, Quantification of CSF cells shows that the majority of cells are T cells, with a minority population of CD14⁺ monocytes and a quantifiably minor number of CD19⁺ B cells (n = 10 healthy subjects); mean ± s.e.m. c, Quantification of CD8⁺ T cell subsets shows that the majority of cells are T_EM or T_EMRA cells (n = 10 healthy subjects); mean ± s.e.m. d, Single-cell sorting of CSF cells shows that CD4 and CD8 T cells were sorted from control individuals and patients with AD. Data were replicated in two independent experiments. e, Donut plots depicting CSF clonality in plate-seq samples. Clones are coloured by their proportion of the total TCRαβ sequences for each subject. f, Gating strategy for drop-seq experiments. Live CSF cells were sorted using a live/dead marker. g, Multidimensional reduction and visualization by t-SNE shows distribution of CSF cells by group, sex and patient (n = 9 healthy, n = 9 MCI or AD).

**Extended Data Fig. 9 |. Clonally expanded CD8⁺ T cells in CSF from patients with AD and patients with PD.**
a, The top individual clones in AD were assessed by combining scRNA-seq and scTCR-seq datasets. A MAST differential expression test with Benjamini-Hochberg correction was conducted to compare CD8⁺ T cell maximum clones (n = 12–18 cells) from patients with MCI or AD against all CSF T cells. Note the colocalization ofAD clones with CD8⁺ clusters on the t-SNE plots. b, Separate analysis of patients with MCI, AD and PD by percentages of maximum clones revealed an enrichment of highly expanded clones (defined as comprising 3% or more of all TCRαβ sequences; indicated by dotted line) in patients with these diseases. Only one out of ten healthy subjects had a highly expanded clone in their CSF, versus four out of six patients with AD, two out of six patients with PD and one out of five patients with MCI. c, Quantification of overall clonality (defined as the percentage of total TCRαβ sequences that are identical to one or more TCRαβ sequences) in the four groups of cohort 4. Significance was measured by two-way ANOVA followed by Tukey’s multiple comparisons test. Only samples with detectable clones were included in the analysis (n = 11 healthy; n = 5 MCI; n = 5 AD; n = 6 PD). Box plots in b, c Box plots show median and 25th to 75th percentiles, and whiskers indicate the minimum and maximum values. d, Gene-expression analysis was conducted on all 24 samples and clustered by t-SNE. Clusters were composed of a mixture of groups, patients, clonal and non-clonal cells. e, Genes (encoding cytotoxic effector proteins) that showed increased expression in a maximum PD clone (n = 14 cells) were analogous to those observed to be overexpressed in AD clones. MAST differential expression test with Benjamini-Hochberg correction.

**Extended Data Fig. 10 |. EBV BZLF1 antigen identification in the CSF of patients with AD.**
a, TCRβ chains derived from GLIPH were used to clone two full TCRαβ TCRs that were introduced into TCR-deficient SKW-3 cells by lentiviral transduction. TCRαβ 1 and TCRαβ 2 cell lines expressed TCRαβ by flow cytometry but controls (no virus and empty lenti viral vector) did not express TCRαβ. Data were replicated in three independent experiments. b, Both TCRαβ 1 and TCRαβ 2 cells upregulate the activation marker CD69 after stimulation with αCD2/CD3/CD28 beads (n = 3 per group). One-way ANOVA (F(3,8) = 204.02, P = 6.78 × 10⁻⁸) with Tukey’s test for multiple comparisons; mean ± s.e.m. c, Gating strategy for MHC-I peptide pool experiments. d, TCRαβ 1 and TCRαβ 2 were presented with antigens in a non-autologous fashion (mismatch between fibroblast and TCR). No significant differences in reactivity were detected in either cell line. One-way ANOVA (F(3,8) = 1.16,P = 0.38) with Tukey’s test for multiple comparisons; mean ± s.e.m. e, Individual histograms of autologous candidate peptide stimulations. CD69 expression is shown for control DMSO and each peptide for both cell lines. Note the increased expression of CD69 induced by peptide 7 in TCRαβ 1 cells. Data were replicated in three independent experiments. f, Gating strategy for quantifying HLA-B*08:01 EBV BZLF1 dextramer positivity.

**Fig. 1 |. Peripheral CD8⁺ T_EMRA cells are increased in AD and are negatively associated with cognition.**
a, Four cohorts were used to assess adaptive immunity in AD. b, Representative SPADE trees of PBMCs from healthy individuals and patients with MCI or AD in cohort 1 show an increased abundance of a CD8⁺ cluster (cluster 63) in patients with MCI or AD. Background tree nodes are sized according to cell counts. Insets are coloured according to CD8 expression. c, Quantification of cluster 63 as a percentage of total PBMCs. The percentage of cluster 63 cells is significantly higher in patients with MCI or AD than healthy control individuals. Mean ± s.e.m.; unpaired two-sided t-test with Welch’s correction. d, Marker expression analysis of cluster 63 corresponds to a CD3⁺CD8⁺CD45RA⁺CD27⁻ T_EMRA population. Data in **c, d** were pooled from seven independent experiments with similar results. e, Linear regression showing the inverse correlation between cognitive score and the percentage of CD8⁺ T_EMRA cells in individuals from cohort 2. Pearson’s correlation r values are shown for each group. The significance of the difference between the two data sets was measured by ANCOVA. f, Stimulation with PMA and ionomycin (stim.) induces increased expression of IFN-γ in CD8⁺ T cells from patients with MCI or AD. Mean ± s.e.m.; unpaired two-sided t-test with Welch’s correction. g, Differential expression analysis (scRNA-seq) of CD8⁺ T_emra cells from healthy individuals (n = 7) and patients with MCI or AD (n = 6) shows upregulated TCR signalling. Model-based analysis of single-cell transcriptomics (MAST) differential expression test with Benjamini-Hochberg correction. h, Pathway analysis of differentially expressed genes in CD8⁺ T_EMRA cells from patients with MCI or AD (n = 6 subjects) versus healthy individuals (n = 7 subjects) shows increased antigenic stimulation of CD8⁺ T_EMRA cells in patients with MCI or AD. Fisher’s exact test with Benjamini-Hochberg correction. Pathways (circles) with positive z-scores are coloured red; those with negative z-scores are coloured blue. The size of the circle corresponds to the size of the z -score (two-sided).

**Fig. 2 |. CD8⁺ T cells enter the brain in patients with AD.**
a, Confocal imaging of cerebral amyloid angiopathy (CAA) in the post-mortem brain of a patient with AD from cohort 3 shows CD8⁺ T cells in the perivascular space of Aβ⁺ blood vessels with cerebral amyloid angiopathy in three AD-affected hippocampi. Arrowheads indicate CD8⁺ T cells; asterisks indicate blood vessel lumen. Scale bars, 20 μm. b, Higher numbers of CD8⁺ T cells were detected in AD-affected than control hippocampi. Mean ± s.e.m.; unpaired two-sided t-test with Welch’s correction. c, A CD8⁺ T cell is shown associated with MAP2⁺ neuronal processes. d, CD8⁺ T cells are localized to the leptomeninges and adjacent to hippocampal AP plaques. Scale bar, 100 μm. Data in c, d were replicated in three independent experiments.

**Fig. 3 |. Clonal expansion of CD8⁺ T_EMRA cells in the CSF of patients with AD.**
a, Plate-seq and drop-seq methods used for scTCR-seq and scRNA-seq of immune cells of the CSF in patients from cohort 4. b, CD8⁺ TCRαβ clonality (plate-seq) in the CSF of patients with AD and healthy control individuals. c, The top (most expanded) clone in AD had a marker expression profile of CD8⁺CD45RA⁺CD27⁻ T_EMRA cells. Data were replicated in two independent experiments. d, CSF cells analysed by drop-seq and clustered by multidimensional reduction with t-SNE, showing populations of immune cells that include CD8⁺ T_EMRA cells (n = 9 healthy control individuals (10,876 cells); n = 9 patients with MCI or AD (10,391 cells)). e, Marker expression of CSF clusters, including CD8+ T_EMRA cells. *CD62L* is also known as *SELL; CD11c* is also known as *ITGAX*. Data were pooled from three independent experiments. f, Concentration of clonal cells in locations of CD8⁺ T cell clusters (n = 9 subjects per group). g, Representative plots of CD8⁺ TCRαβ clonality (drop-seq) in age-matched subjects shows enhanced clonal expansion and more highly expanded clones in AD. Clones are coloured by proportion of the total TCRαβ sequences. h, Quantification of maximum clones (% TCRαβ sequences) shows a higher percentage in patients with MCI or AD than healthy control individuals (n = 9 subjects per group). Samples lacking clonal cells were scored as zero. Box plots show median and 25th to 75th percentiles, and whiskers indicate the minimum and maximum values. Unpaired two-sided f-test with Welch’s correction. i, Differential expression of highly expanded clones (clonal TCRαβ > 5) revealed increased expression of cytotoxic effector genes. MAST differential expression test with Benjamini-Hochberg correction (n = 9 patients with MCI or AD). j , Quantification of highly expanded clones (clonal TCRαβ > 5) showed that 49.13% of them are CD8⁺ T_EMRA cells (n = 9 patients with MCI or AD). k, Increased expression of *B2M, NKG7*and *GZMA* in clonal CD8⁺ T cells from patients with MCI or AD. MAST differential expression test with Benjamini-Hochberg correction (n = 9 subjects/group). l, A hippocampal CD8⁺ T cell in an AD-affected brain (cohort 3) shows expression of GZMA adjacent to MAP2⁺ neuronal processes. Scale bar, 5 pm. Data were replicated in three independent experiments. m, Percentages of CD8⁺ T cells that express GZMA in control and AD-affected hippocampi. Mean ± s.e.m.; unpaired two-sided t-test with Welch’s correction.

**Fig. 4 |. Antigen identification of clonally expanded TCRs in the CSF of patients with AD.**
a, Unweighted network analysis of CD8 T CRαβ sequences combined from plate-seq and drop-seq experiments. Group node IDs with individual TCRαβ clones are depicted as circles and sized according to the proportion of total sequences of each clone. Arrow indicates a shared clonal T CRaP sequence with specificity for EBV EBNA3A. Note that several healthy control (HC) subjects have no clones. b, Shared TCRαβ sequences among patients with MCI or AD. Three patients had identical TCRβ chains with specificity for EBV EBNA3A. The antigen specificity of T CRβ is shown below. c, Differential expression of EBV-specific clones in MCI and AD (from n = 3 subjects) versus all CSF T cells shows enhanced expression of cytotoxic effector genes. MAST differential expression test with Benjamini-Hochberg correction. d, Workflow for antigen identification of CSF TCRs. GLIPH was applied to TCR sequencing to derive homologous TCR sequences between patients. GLIPH identified two patients with AD who had identical TCRβ chains and a third patient with a similar sequence. The T CRαβ sequences derived from GLIPH were introduced into SKW-3 cells. e, Autologous fibroblasts were used to present antigens to TCRαβ 1 and TCRαβ 2 cells. Only TCRαβ 1 cells showed significant upregulation of CD69 following antigen presentation. Data are averages from three separate experiments performed in triplicate.Mean ± s.e.m.; one-way analysis of variance (ANOVA) (F(3, 8) = 1,050,P = 1.01 × 10⁻¹⁰) with Tukey’s multiple comparisons test. f, Peptide 7 (RAKFKQLL) of the EBV trans-activator BZLF1 protein activates TCRαβ 1 but not TCRαβ 2 cells. Two-way ANOVA (F(14, 60) = 14.06, P =4.8 × 10⁻¹⁴) followed by Sidak’s multiple comparisons test. Data were pooled from n = 3 independent experiments. The P value shown is from comparing peptide 7 values for each cell line. Mean ± s.d. g, A fluorescent dextramer composed of H LA-B*08:01 presenting the BZLF1 peptide RAKFKQLL shows nearly 100% positivity with TCRαβ 1 but no positivity with TCRαβ 2 cells. Unpaired two-sided t-test with Welch’s correction (n = 6 per group).

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Comment in

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Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer's disease

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Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer's disease

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