背景:越来越多的证据支持适应性免疫参与放射性脑损伤(RIBI)的发展。我们先前的工作强调了RIBI中CD8+T细胞的细胞毒性功能。在这项研究中,我们旨在研究RIBI中细胞毒性CD4+T细胞(CD4+CTLs)的存在和潜在作用,以更全面地了解这种情况下的适应性免疫.
方法:利用单细胞RNA测序(scRNA-seq),我们分析了4例RIBI患者脑部病变的3934个CD4+T细胞,并确定了该人群中的6个亚簇.一个值得注意的子集,细胞毒性CD4+T细胞(CD4+CTLs),具有高表达的细胞毒性相关基因(NKG7,GZMH,GNLY,FGFBP2和GZMB)和几种趋化因子和趋化因子受体(CCL5,CX3CR1和CCL4L2)。通过深入的伪时间分析,模拟CD4+T细胞的发育,我们观察到CD4+CTL表现出终末分化特征.它们的功能富含蛋白质丝氨酸/苏氨酸激酶活性,GTP酶调节活性,磷蛋白磷酸酶活性,和半胱氨酸型内肽酶活性参与凋亡信号通路。相应地,接受伽玛刀照射的小鼠大脑显示CD4+T细胞的时间依赖性浸润,MHCII+细胞的增加,以及病变中CD4+CTLs的存在,伴随着凋亡相关蛋白的升高。最后,最重要的是,患者水平的单细胞T细胞受体测序(scTCR-seq)分析确定了RIBI病变组织中CD4+CTL的大量克隆扩增。转录因子编码基因TBX21,RORB,EOMES与CD4+T细胞的细胞毒功能呈正相关,提示他们区分RIBI相关CD4+CTLs与其他亚群的潜力。
结论:本研究丰富了对RIBI患者适应性免疫细胞转录景观的理解。它提供了RIBI病变中克隆扩增的CD4+CTL亚群的首次描述,这可能阐明了RIBI发展的新机制,并为该疾病提供了潜在的生物标志物或治疗靶标。
Accumulating evidence supports the involvement of adaptive immunity in the development of radiation-induced brain injury (RIBI). Our previous work has emphasized the cytotoxic function of CD8+ T cells in RIBI. In this study, we aimed to investigate the presence and potential roles of cytotoxic CD4+ T cells (CD4+ CTLs) in RIBI to gain a more comprehensive understanding of adaptive immunity in this context.
Utilizing single-cell RNA sequencing (scRNA-seq), we analyzed 3934 CD4+ T cells from the brain lesions of four RIBI patients and identified six subclusters within this population. A notable subset, the cytotoxic CD4+ T cells (CD4+ CTLs), was marked with high expression of cytotoxicity-related genes (NKG7, GZMH, GNLY, FGFBP2, and GZMB) and several chemokine and chemokine receptors (CCL5, CX3CR1, and CCL4L2). Through in-depth pseudotime analysis, which simulates the development of CD4+ T cells, we observed that the CD4+ CTLs exhibited signatures of terminal differentiation. Their functions were enriched in protein serine/threonine kinase activity, GTPase regulator activity, phosphoprotein phosphatase activity, and cysteine-type endopeptidase activity involved in the apoptotic signaling pathway. Correspondingly, mice subjected to gamma knife irradiation on the brain showed a time-dependent infiltration of CD4+ T cells, an increase of MHCII+ cells, and the existence of CD4+ CTLs in lesions, along with an elevation of apoptotic-related proteins. Finally, and most crucially, single-cell T-cell receptor sequencing (scTCR-seq) analysis at the patient level determined a large clonal expansion of CD4+ CTLs in lesion tissues of RIBI. Transcriptional factor-encoding genes TBX21, RORB, and EOMES showed positive correlations with the cytotoxic functions of CD4+ T cells, suggesting their potential to distinguish RIBI-related CD4+ CTLs from other subsets.
The present study enriches the understanding of the transcriptional landscape of adaptive immune cells in RIBI patients. It provides the first description of a clonally expanded CD4+ CTL subset in RIBI lesions, which may illuminate new mechanisms in the development of RIBI and offer potential biomarkers or therapeutic targets for the disease.