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Single cell transcriptome combined with TCR|| in the cerebrospinal fluid of Alzheimer's patients has cloned CD8+T cells. Time: 2020-02-27

Neuroinflammation in Alzheimer's disease ( Alzheimer 's disease , Abbreviation AD ) It plays a crucial role, but for the adaptive immune response and AD Between relationship Little is known about it. The lymphatic system of the brain sends the immune cells from the cerebrospinal fluid (CSF) to the deep cervical lymph nodes, so that peripheral T cells can respond to the brain antigens. However, AD Patient continuity Whether neuroinflammation is related to the entry of T cells into the brain is not yet known.

T cell receptor ( T cell receptor The interaction between TCR and antigen is very important for adaptive immunity. When T cells recognize homologous antigens (cognate antigen), they will be cloned. Moreover, TCR sequences are diverse, and have specific TCR sequences for single T cells. Therefore, it is found that two or more T cells with the same TCR sequence can be considered as evidence of clonal expansion of T cells. At present, studies have found that the distribution and function of T cells in peripheral blood of AD patients and the changes of cytokines secretion, but the antigens causing these changes are still unclear.


Two thousand and twenty year One 8, Stanford University Mark M Davis and Tony Wyss-Coray team find stay AD Cloned T cells were found in cerebrospinal fluid of patients. One category is also found. T Cells are related to cognitive ability through single cells. TCR It is found that the T cells can be identified by cloning. EB Two different antigens of the virus. The research Research results Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer "fluid" Publish on topic stay Nature In magazines.

Analytical thinking:


Sample information:

PBMC, brain tissue, cerebrospinal fluid

Detection platform:

Mass spectrometry, flow cytometry, immunohistochemistry, Plate-seq, Drop-seq (10X Genomics)

Research findings:

One AD Peripheral blood of patients CD8 + T EMRA The number of cells is negatively correlated with cognitive ability.

The researchers assessed the adaptive immunity of AD by using multiple cohort analyses (Figure 1a). The peripheral blood mononuclear cells (PBMC) of AD patients and mild cognitive impairment (MCI) patients were studied by mass spectrometry flow cytometry. Then spanning-tree progression analysis of density-normalized events (SPADE) was used to cluster analysis. It was found that patients with MCI or AD had significantly increased number of CD8. + Population (Fig. 1b), that is, cluster 63. The marker of cell population and CD3 + CD8 + CD27 - T effect memory CD45RA + (T EMRA Cell markers are identical (Fig. 1D). They are a group of effector cells that secrete proinflammatory cytokines and cytotoxic molecules.

In order to further study CD8 + The role of T cells in MCI and AD is studied by Cohort 2 In Figure 1a, the relationship between cognitive and memory T cell populations was assessed. It is revealed that in MCI and AD, CD8 + TEMRA cells were negatively correlated with cognitive ability (1E). After PMA (phorbol 12-myristate 13-acetate) was used to stimulate PBMC, CD8 was obtained from MCI or AD patients. + The level of IFN- gamma in T cells was significantly higher than that in control CD8 + T cells (Fig. 1F), peripheral CD8 of patients with MCI or AD. + T cells have some functional differences compared with normal persons. Then on CD8 + TEMRA cells carry out drop-seq single cell sequencing, and differential expression and pathway analysis revealed that CD8 was found in MCI and AD patients. + The amino acid metabolism of TEMRA cells was significantly reduced, and TCR and cytokine signaling increased (1g, H).

The above results showed that compared with the control group, MCI and AD Peripheral blood of patients CD8 + TEMRA The cells have greater antigenic irritation.


Figure 1. TEMRA cells found in MCI/AD patients are more stimulated by antigen.

Two CD8 + T Cell entry AD Patient's brain

Next, researchers want to identify Is there a CD8 in the brain of AD patients? + T cells carry out CD8 and A beta in the brain of normal persons and AD patients in Cohort 3 (Fig. 1a). Immunohistochemical assay And CD8 + Analysis of the proximity of T cells to cerebral vessels. A large number of extravascular CD8 were found in the perivascular spaces of three AD brain hippocampus with amyloid angiopathy. + T cells (Fig. 2a), subject to AD affects CD3 in hippocampus. + CD8 + The number of T cells was significantly higher than that of the control hippocampus (2b), and CD8 + T cells and microtubule associated protein 2 (MAP2) + Neurons have a certain correlation (Fig. 2C). Finally, researchers also detected CD3 in the pia near the hippocampus of AD patients. + CD8 + T cells (Fig. 2D).

The above results show that AD The patient's brain was detected. CD8 + T Cell survival Yes.


Fig. 2 CD8 was detected in the brains of AD patients. + T cell


Three AD Cerebrospinal fluid in patients CD8 + TEMRA Cell cloning and amplification

CD8 + The location of T cells in the pia of the brain has prompted researchers to continue to explore whether antigen-specific cells exist in CSF. The researchers found that the immune cells in the CSF of healthy elderly subjects mainly contained T cells, and a small number of innate immune cells and trace B cells, of which CD8 + T cells are almost entirely effector memory T cells (T effector memory, T). EM The cells were composed of TEMRA cells, which accounted for about T EM 20% of the group.

In the following study, researchers used Two sequencing methods of plate-seq and drop-seq were carried out. T cell cloning and amplification assessment, patient data are derived from Cohort 4 (Figure 1a). It is found that the T cells in CSF are mainly CD8. + T cells were cloned and a large number of clones were found in one AD patient. Accounting for CD8 + 44% of T cells (Fig. 3b). Subsequently, the Marker gene was identified as the clone. CD8 + CD45RA + CD27 - TEMRA cells (Fig. 3C). The discovery was first put forward. Cloned amplification is present in cerebrospinal fluid of the brain affected by age-related neurodegenerative diseases. CD8 + T Cells.

When researchers used drop-seq to divide cells into CSF cells, it was also found that most CSF cells were T cells (Map 3D, e). These cells were then subjected to single cell TCR sequencing (scTCR-seq) and found that there were many highly amplified CD8 in CSF cells from patients with MCI or AD. + In contrast, the control group was the opposite (Fig. 3F, G, H). Differential expression analysis revealed that the highly amplified clones in MCI and AD (that is, T cells with more than five TCR alpha beta sequences) showed increased expression of cytotoxic effect genes (Fig. 3I). adopt The proportion analysis showed that 49.13% of the cloned cells were CD8. + TEMRA cells (Fig. 3j), and these cells in MCI and AD also showed increased expression of cytotoxic effect genes. Especially in MCI and AD patients, high expression affects the senescence factor of cognition, human leukocyte antigen C (HLA-C) and beta -2- microglobulin (B2M), and other AD related genes. (Fig. 3K). The researchers also found that CD8 + Expression of GZMA gene in T cells (Fig. 3L) and GZMA in hippocampus of AD patients + CD8 + Cells have a higher percentage (Fig. 3M).

The above results reveal that AD Patients CSF in CD8 + TEMRA The proinflammatory and cytotoxic functions of cloned cells.

chart Three   AD Patients CSF in CD8 + TEMRA The cells are cloned and have proinflammatory functions.

4.AD Cloning and amplification of cerebrospinal fluid in patients TCRs Antigen identification

Next, the researchers hope to determine which antigen causes it. CD8 + TEMRA Cell cloning and amplification. By constructing TCR alpha beta Sequential An unweighted network found a shared clone between one case of MCI and one patient with AD, while the other AD patient shared one of them. TCR beta Sequence, this one. TCR beta sequence ( CASSLGQAYEQYF ) Yes A herpesvirus EBV EBNA3A Antigen ( Herpesviridae Epstein – Barr nuclear antigen 3 ) It is specific. 4B ) and The The cytotoxic effect gene expression was increased in clone. 4C ) Subsequently, researchers used GLIPH Algorithm pair Unknown TCR Sequence Specific antigen The determination. find AD Patients shared TCR beta chain ( CASSLAGGYNEQFF And Third patients with AD. TCR beta Chain ( CASSLGTGNNEQFF ) Homology (map) 4d )

By constructing expression TCR alpha beta 1 and TCR alpha beta 2 Cell lines, found TCR alpha beta 1 Cell pair MHC-1 The peptide library is responsive. TCR alpha beta 2 No cell response (Fig. 4e ) Then will TCR alpha beta 2 As a comparison, only one peptide was activated. TCR alpha beta 1 Cells, that is, EB virus transactivator protein BZLF1 Of 7-RAKFKQL L (Fig. 4f, G )

Above, through sequencing and analyzing TCR, researchers found a new type of TCR in AD, which is specific for EBV transactivator protein BZLF1.

chart Four   AD Patient CSF In cell TCR Antigen identification

To sum up, the researchers passed the test. TCR The sequence analysis proves that AD in CD8 + T Amplification and identification of the antigen specific clones of the cells. EBV Antigen specificity TCR Though not enough to explain EBV Infection and AD There is a clear causal relationship, but through analysis TCR Identification, cloning and adoption TCR Identification of antigens will provide a new way to study the adaptive immunity of neurodegenerative diseases.


Link: Http://nature.webvpn.ustc.edu.cn/articles/s41586-019-1895-7


Before sorting out a series of single cell transcriptome sequencing data, we analyzed the content of the related elements, the tools, and the results of the analysis. Please mark the links below. Oh, strong ice organisms can not only carry out professional single cell transcriptome sequencing, but also formally carry out single cell TCR-Seq. If necessary, welcome to consult!

Single cell transcriptome sequencing data analysis course

(1) five kernels? Yolk? BD Rhapsody? 10 x Genomics? How to select the sorting platform?

2. @ strong ice organisms, please give me a single cell transcriptome sequencing data analysis strategy.

(3) scRNA-Seq data analysis.

(4) analysis of ScRNA-Seq|| single cell transcriptome data by quasi sequential analysis

SCENIC analysis of single cell transcriptome data analysis -- searching for the driving genes in cell population


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