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摘要
• 摘要:   目的  基于单细胞RNA测序（single cell RNA sequencing, scRNA-seq）挖掘阿尔茨海默病（Alzheimer's disease, AD）外周血免疫特征作为生物标志物，系统性探索AD外周血免疫细胞亚型丰度、基因表达特征和细胞通讯异常。  方法  从GEO数据库中下载AD外周血免疫细胞scRNA-seq数据集GSE168522，于RAD-Blood网页服务器（//www.bioinform.cn/RAD-Blood/）中分析AD患者血液细胞组成成分变化，利用CellChat分析AD患者血液中异常的细胞间通讯作用。  结果  AD患者和健康人血液中有两种CD8⁺ T细胞，其中一类高表达颗粒酶K（granzyme K, GZMK）〔伪发现率（false discovery rate, FDR）<0.05〕，另一类高表达GZMA、GZMB和GZMH（FDR<0.05）。GZMK⁺ CD8⁺ T细胞在AD患者血液中含量升高32.9%（P=5.15E-21），与其他细胞类型的交互作用增加，并可能通过主要组织相容性复合体Ⅰ类（major histocompatibility complex class Ⅰ, MHC-Ⅰ）信号转导异常与AD关联，红细胞为GZMK⁺ CD8⁺ T细胞MHC-Ⅰ信号通路异常提供了主要配体，即人类白细胞抗原（human leukocyte antigen, HLA）Ⅰ类分子（HLA-A、HLA-B、HLA-C和HLA-E）。血液RESISTIN信号通路仅富集于AD患者血液中，其可能是AD血液特异性信号通路。  结论  外周血GZMK⁺ CD8⁺ T细胞含量升高、GZMK⁺ CD8⁺ T细胞与红细胞的交互作用增加、RESISTIN通路增强是潜在的AD标志物。

  关键词:
  • 阿尔茨海默病 / 
  • 单细胞RNA测序 / 
  • koko体育app: 血液生物标志物 / 
  • koko体育app: CD8⁺ T细胞 
  Abstract:   Objective  Based on single-cell RNA sequencing (scRNA-seq) to explore immune characteristics in the peripheral blood of patients with Alzheimer's disease (AD) as biomarkers.  Methods  GSE168522, the scRNA-seq dataset of AD peripheral blood immune cells, was downloaded from the Gene Expression Omnibus (GEO) database and was analyzed in the RAD-Blood web server (//www.bioinform.cn/RAD-Blood/). The changes in blood cell composition in AD patients were analyzed. The abnormal communications between different types of cells in AD patients were investigated by the CellChat R package.   Results  There were two kinds of CD8⁺ T cells in the blood of AD patients and healthy individuals, one of which highly expressed granzyme K (GZMK) (false discovery rate [FDR]<0.05), and the other highly expressed GZMA, GZMB, and GZMH (FDR<0.05). In the blood of AD patients, the content of GZMK⁺ CD8⁺ T cells was increased by 32.9% (P=5.15E-21), their interactions with other cell types were increased, and they might be associated with AD through the abnormal signal transduction of major histocompatibility complex class Ⅰ (MHC-Ⅰ). Erythrocyte provided the main ligands, that are, human leukocyte antigen (HLA) class Ⅰ molecules, including HLA-A, HLA-B, HLA-C, and HLA-E, for the abnormal MHC-Ⅰ signaling pathway of GZMK⁺ CD8⁺ T cells. The RESISTIN signaling pathway was specifically enriched in the blood of AD patients.  Conclusion  The increased content of peripheral blood GZMK⁺ CD8⁺ T cells, the increased interaction between GZMK⁺ CD8⁺ T cells and erythrocytes, and the enhanced RESISTIN pathway are potential blood biomarkers of AD.

  Key words:
  • Alzheimer's disease / 
  • scRNA-seq / 
  • Blood biomarkers / 
  • CD8⁺ T cell 
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  图  1  细胞类型分布情况

  Figure  1.  Distribution of the cell types

  ^* P<0.05.

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  图  2  细胞间基因表达差异

  Figure  2.  Differences in gene exꦅpression between cells

  Difference in gene expression between B cells and CD8⁺ T cells and other cell types. T༺he size of tꦚhe bubbles represents the fraction of genes in the subgroup; the shades of the color indicate the level of gene expression, with darker colors indicating higher gene expression.

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  图  3  AD患者和健康人血液中细胞交互数目及强度差异

  Figure  3.  Diffe🔯ren꧂ces in the number and strength of cell interactions between AD patients and controls

  A, Bubble plots of the number (left) and strength (right) of cellular interactions in GZMK⁺ CD8⁺ T cell. B, GZMK⁺ CD8⁺ T cell as signal sender (left) and receiver (right) versus other cell interactions between AD patients and controls. Red (or blue) arrows indicate increas♒ed (or decreased) signals in AD patients compared with controls. C, The differences in the strength of inter-cellular communication between AD patients and controls. The colored bar charts at the top (or right) represent the sum of absolute values ꦫof the column values (or row values) shown in the heat map. The red (or blue) in each cell indicates an increased (or decreased) signal in AD patients compared to controls.

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  图  4  AD患者和健康人的血液免疫细胞通讯网络中的信号通路信息流差异

  Figure  4.  Difference in information flow of signal pathway in cellular communication network between AD patient and control blood s꧅amples

  A, The bar chart shows the difference in overall information flow between AD patients and controls. The red signal pathway is enriched in AD patients, while the green signal pathway is enriched in healthy controls. The font color on the ordinate represents the significance of the Wilcox test, with red and green representing P<0.05 and black representing P>0.05. B, The overall signal pattern in AD patients and controls. The top (or right) bar graph represents the sum of column values (or row values) displayed in the heat map and the darker the color, the higher the signal strength. C, Statistical of the incoming (left) and outgoing (right) signal patterns of GZMK⁺ CD8⁺ T cell in AD patients and controls.

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  图  5  AD患者和健康人血液中GZMK⁺ CD8⁺ T细胞与其他细胞间配体-受体对变化差异

  Figure  5.  Differences in changes between AD patients and controls in intercellular ligand-receptor pairs between GZMK⁺ CD8⁺ T cell and other cells

  A, Probability of communication mediated by ligand-receptor pairs from other cell types to GZMK⁺ CD8⁺ T cell; B, probability of communication mediated by ligand-receptor pairs from GZMK⁺ CD8⁺ T cell to other cell types.

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  图  6  MHC-Ⅰ通路的配体和受体基因的表达情况

  Figure  6.  Expression of ligand and receptor genes of MHC꧂-Ⅰ pathway

  A, Expression difference of CD8A and CD8B genes between AD patients and controls in GZMK⁺ CD8⁺ T cell; B, expression difference of HLA-A, HLA-B, HLA-C, and HLA-E genes in erythrocyte between AD patients and controls. The abbreviation avg_log₂FC represents average log₂ fold change.

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  表  1  鉴定细胞类型的标记基因

  Table  1.   Marker 𒐪genes used for cell type identification

  Cell type	Marker genes
  B cell	CD19	MS4A1	CD79A	CD79B	CD27
  CD4+ T cell	IL7R	GZMK	KLRB1	KLRG1	CD3D
  CD8+ T cell	CD8A	CD8B	CD3D	CD3E	CD3G
  Dendritic cell	FCER1A	CD1C	CLEC10A	HLA-DQA1	CST3
  Erythrocyte	ALAS2	HBA1	HBA2	HBB	GYPA
  Megakaryocyte	PPBP	PF4	GP9	MYL9	TUBB1
  Monocyte	S100A8	LYZ	S100A9	S100A12	AIF1	VCAN	FCN1
  Naive CD8+ T cell	LDHB	GAS5	CCR7	LEF1	TCF7
  Natural killer cell	TRDC	FGFBP2	NKG7	GNLY	KLRF1
  Plasmacytoid dendritic cell	LILRA4	PLD4	STMN1	TXN	PARK7	NUCB2

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  表  2  细胞分布差异分析（卡方检验）

  Table  2.   🗹 Cell distribution difference analysis 𓄧(Chi-square test)

  Cell	AD (cell number)	Control (cell number)	χ2	df	P
  B cell_1	1563	629	0.29	1	0.59
  B cell_2	1052	473	6.19	1	0.01
  B cell_3	102	11	19.03	1	1.28E-05
  CD4+ T cell	1830	250	277.14	1	3.15E-62
  CD8+ T cell_1	5079	2083	3.22	1	0.07
  CD8+ T cell_2	4333	1278	88.47	1	5.15E-21
  Dendritic cell	385	141	0.50	1	0.48
  Erythrocyte	172	270	237.99	1	1.08E-53
  Megakaryocyte_1	1569	765	25.21	1	5.14E-07
  Megakaryocyte_2	417	418	199.94	1	2.15E-45
  Monocyte_1	7941	2887	14.77	1	1.21E-04
  Monocyte_2	2139	818	0.41	1	0.52
  Monocyte_3	420	316	80.01	1	3.72E-19
  Monocyte_4	90	25	2.36	1	0.12
  Naive CD8+ T cell_1	4730	2061	17.52	1	2.84E-05
  Naive CD8+ T cell_2	3748	1792	51.74	1	6.34E-13
  Naive CD8+ T cell_3	695	203	13.99	1	1.84E-04
  Natural killer cell_1	7639	2717	22.97	1	1.65E-06
  Natural killer cell_2	3604	1523	6.36	1	0.01
  Plasmacytoid dendritic cell_1	238	85	0.56	1	0.45
  Plasmacytoid dendritic cell_2	173	60	0.68	1	0.41

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  表  3  CD8⁺ T细胞中AD患者和健康人差异基因表达情况

  Table  3.   DEGs in CD8⁺ T cells between AD patients and controls

  Gene	CD8+ T cell_1			CD8+ T cell_2
  	Marker	AD vs. Control		Marker	AD vs. Control
  GZMA	Yes	Up		No	Up
  GZMB	Yes	Down		No	Up (Not significant)
  GZMH	Yes	Up		No	Up
  GZMK	No	Up		Yes	Down (Not significant)
  LTB	No	Up (Not significant)		Yes	Down
  CMC1	No	Up		Yes	Up
  CD74	No	Up		Yes	Up
  PRF1	Yes	Down (Not significant)		No	Up
  GNLY	Yes	Down (Not significant)		No	Down (Not significant)
  Up represents log2 fold change>0 and FDR<0.05; Down represents log2 fold change<0 and FDR<0.05; Not significance represents FDR>0.05.

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