Alloantigen Infusion Selectively Activates the Transcriptome of Type 2 Conventional Dendritic Cells"

Recent studies have revealed novel molecular mechanisms by which innate monocytic cells acutely recognize and respond to alloantigen, and the significance of this to allograft rejection and immunologic tolerance. What remains unclear is the single cell heterogeneity of the innate allo-response, and particularly the contribution of dendritic cell (DC) subsets. To investigate the acute response of innate immune cells and DC subsets to exposure of alloantigen, experimental rodents were administered an intravenous injection of live allogenic cells versus isogenic cells. In parallel, we also infused apoptotic allogenic and isogenic cells, which have been reported to modulate immunity. Forty-eight hours after injection, recipient spleens were harvested, pooled by condition, enriched for DCs, and subjected to single cell mRNA sequencing and downstream analysis. Injection of live splenic cells induced a greater quantity of transcriptional changes across all DC subsets as identified by differential gene expression analysis and compared to apoptotic cell injection. In the setting of live cell infusion, type 2 conventional dendritic cells (cDC2s) were more transcriptionally responsive with a Ccr2+ cDC2 subcluster uniquely responding to the presence of alloantigen compared to isogenic control. Candidate receptors of allorecognition in other innate populations were interrogated amongst cDC2s and A-type paired immunoglobulin-like receptors (PIR-As) was found to be increased specifically in the cDC2 population following allo-exposure at the mRNA level and by flow cytometry. These results illuminate previously unclear distinctions between therapeutic infusions of live versus apoptotic allogenic cells and newly implicate cDC2 cells in innate allorecognition in the context of transplantation. Splenic cells magnetically enriched for DCs and CD45+ cells from mice infused with live allo- (LA, Balbc), live iso- (LI, B6), apopototic allo- (AA, Balbc), apoptotic iso- (AI, B6) antigen or saline control (C) 48 hours prior and subjected to single cell RNA sequencing.

近期研究揭示了先天单核细胞急性识别并应答同种异体抗原的新型分子机制，以及该机制在同种异体移植排斥与免疫耐受中的重要意义。目前尚未明确的是先天同种应答的单细胞异质性，尤其是树突状细胞（dendritic cell, DC）亚群所发挥的作用。为探究先天免疫细胞及DC亚群对同种异体抗原暴露的急性应答反应，本研究对实验啮齿类动物分别静脉注射活的同种异体细胞与同基因细胞。与此同时，本研究还输注了据报道可调节免疫功能的凋亡型同种异体及同基因细胞。注射后48小时，收集受体小鼠的脾脏，按实验分组混合后富集DC，随后进行单细胞mRNA测序及下游分析。差异基因表达分析结果显示，与凋亡细胞注射组相比，活的脾脏细胞注射可在所有DC亚群中诱导更多的转录组变化。在活细胞输注的实验条件下，2型常规树突状细胞（conventional dendritic cell type 2, cDC2s）的转录应答更为显著；与同基因对照组相比，Ccr2阳性的cDC2亚簇特异性地对同种异体抗原的存在产生应答。我们在cDC2亚群中筛查了其他先天群体中同种识别的候选受体，结果发现，在mRNA水平及流式细胞术检测中，A型配对免疫球蛋白样受体（A-type paired immunoglobulin-like receptor, PIR-As）在同种异体暴露后的cDC2群体中特异性上调。本研究结果阐明了此前尚未明确的活细胞与凋亡型同种异体细胞治疗性输注之间的差异，并首次揭示了cDC2细胞在移植环境下的先天同种识别过程中发挥的作用。本研究采集了提前48小时接受活同种异体（LA，Balbc品系）、活同基因（LI，B6品系）、凋亡同种异体（AA，Balbc品系）、凋亡同基因（AI，B6品系）抗原输注或生理盐水对照（C）的小鼠脾脏细胞，通过磁珠富集DC与CD45阳性细胞后，进行单细胞RNA测序。