Single cell sequencing reveals the antigen presentation characteristics of dendritic cells and B cells in cardiac grafts
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摘要:
目的 探讨心脏移植物中树突状细胞（DC）和B细胞的抗原提呈特性。 方法 将BALB/c小鼠的心脏移植到C57BL/6J小鼠腹腔内，术后5 d（急性排斥反应早期）提取并流式分选心脏移植物中的CD45+细胞，进行单细胞RNA测序。以心脏移植物中的DC和B细胞的亚群为主要对象，通过生物信息学分析和流式细胞术，研究其在心脏移植后变化趋势、抗原提呈能力及其与T细胞之间的胞间通讯情况。采用基因本体（GO）功能富集差异分析佐证细胞亚群特异性功能和细胞亚群注释可信度。 结果 生发中心样B细胞（GC-L B）是急性排斥反应期心脏移植物中增幅最大、比例高达87%的B细胞亚群，经典DC（cDC）2是心脏移植急性排斥期间唯一大量增多的DC亚群，占44%，是心脏移植后与T细胞的胞间通讯中占据最高通讯强度的DC亚群；单核样DC（moDC）与记忆性B细胞（MBC）是未心脏移植中T细胞输入信号的主要发出者，而在心脏移植后急性排斥反应期中，转变为cDC2与GC-L B；其中MBC与GC-L B分别是心脏移植前后的主要T细胞输入信号来源。 结论 在未移植心脏和移植心脏指向T细胞的胞间通讯中，与DC相比，B细胞均占据更高的通讯数量和权重，推测在心脏移植急性排斥反应早期，B细胞的抗原提呈活动比DC更加活跃，强度更大。 Abstract:Objective To investigate the antigen presentation characteristics of dendritic cells (DC) and B cells in cardiac grafts. Methods The heart of BALB/c mice was transplanted into the abdominal cavity of C57BL/6J mice. CD45+ cells in the heart graft were extracted and sorted by flow cytometry at postoperative 5 d, and single cell RNA sequencing was performed. Taking DC and B cell subsets in cardiac grafts as the main study cells, the changing trend, antigen presenting ability and intercellular communication with T cells after heart transplantation were analyzed by bioinformatics analysis and flow cytometry. Gene ontology (GO) function enrichment difference analysis was adopted to prove the specific function and the reliability annotation of cell subsets. Results Germinal center-like B cell (GC-L B) was the B cell subset with the largest increase in quantity during the acute rejection phase, accounting for 87%. Classical DC (cDC) 2 was the only DC subset with a significant increase in quantity during acute rejection of heart transplantation, accounting for 44% of DC subset, and it occupied the highest communication intensity with T cells after heart transplantation. Mononucleated DC (moDC) and memory B cell (MBC) were the main transmitters of T cell input signals non-transplanted hearts, whereas transformed into cDC2 and GC-L B during the acute rejection phase. Among them, MBC and GC-L B were the main sources of T cell input signals in non-transplanted hearts and heart grafts. Conclusions Compared with DC, B cells occupy a higher number and weight in the intercellular communication with T cells before and after heart transplantation, prompting that the antigen presenting activity of B cells is more active and stronger than DC in the early stage of acute rejection of heart transplantation. -
Key words:
- Heart transplantation /
- Antigen presentation /
- B cell /
- Dendritic cell /
- Single cell sequencing /
- Acute rejection /
- Cellular communication /
- T cell
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图 1 小鼠未移植心脏及心脏移植物中B细胞免疫特征
注：A图为4种B细胞亚型可视化UMAP图；B图为NH组和HTx组4种B细胞亚型数量；C图为Marker基因表达散点UMAP图，横纵坐标代表样本点在低维空间的相对位置关系，颜色深浅代表基因平均表达量高低；D图为代表性差异表达基因小提琴图。
Figure 1. B cell immune characteristics in non-transplanted hearts and heart grafts of mice
图 2 小鼠心脏移植前后DC、B细胞及相应脾脏B细胞流式分析
注：A图为心脏中B细胞流式图（B220+）及统计分析；B图为脾脏中B细胞流式图（CD19+）及统计分析；C图为DC流式图（MHC Ⅱ+、CD11c+）及统计分析。与NH组比较，aP＜0.05。
Figure 2. Flow analysis of DC and B cells and corresponding spleen B cells before and after heart transplantation in mice
图 3 小鼠未移植心脏及心脏移植物中DC免疫特征
注：A图为4种DC亚型可视化UMAP图；B图我为4种DC亚型数量；C图为Marker基因表达散点UMAP图，横纵坐标代表样本点在低维空间的相对位置关系，颜色深浅代表基因平均表达量高低；D图为代表性差异表达基因小提琴图。
Figure 3. DC immune characteristics in non-transplanted hearts and heart grafts of mice
图 4 小鼠心脏DC、B细胞与T细胞胞间交流分析
注：A图为B细胞与T细胞之间胞间通讯的数量和权重可视化图；B图为DC与T细胞之间胞间通讯的数量和权重可视化图；C图为合并DC与B细胞，比对NH组与HTx组DC、B细胞与T细胞之间通讯强度的变化。
Figure 4. Analysis of intercellular communication among DC, B cells and T cells in mouse hearts
图 5 CXCL信号通路在小鼠未移植心脏中DC、B细胞与T细胞之间的胞间通讯分析
注：A图为DC、B细胞与T细胞输入、输出信号分析热图；B图为CXCL信号通路通讯弦图（指向T细胞）；C图为CXCL信号通路通讯角色分析热图；D图为CXCL信号通路中受配体贡献度分析；E图为CXCL信号通路相关基因表达水平小提琴图。
Figure 5. Analysis of intercellular communication between DC, B cells and T cells of CXCL signaling pathway in non-transplanted mouse hearts
图 6 CXCL信号通路在小鼠心脏移植急性排斥反应早期DC、B细胞与T细胞之间的胞间通讯分析
注：A图为DC、B细胞与T细胞输入、输出信号分析热图；B图为CXCL信号通路通讯弦图（指向T细胞）；C图为CXCL信号通路通讯角色分析热图；D图CXCL信号通路中受配体贡献度分析；E图为CXCL信号通路相关基因表达水平小提琴图。
Figure 6. Analysis of intracellular communication among DC, B cells and T cells by CXCL signaling pathway in the early stage of acute rejection of mouse heart transplantation
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