Multi-omics analysis identifies progenitors of CD4-CTLs [scATAC-seq ex vivo]

The CD4+ T cells expressing cytolytic molecules such as granzymes and perforins have been detected in many diseases and are shown to be enriched in the effector memory expressing CD45RA (TEMRA) in humans, but their origin remains elusive. However, their gene expression profile in comparison to classically defined cytotoxic T lymphocytes (CTLs), the CD8-CTLs has not been well demonstrated, hence their relevance remains debatable. Thus in this study by parallelly analysing the CD4-CTLs and CD8-CTLs, we demonstrate that they are indistinguishable for the cytolytic program with both showing similar gene expression profile and T cell antigen-receptor (TCR) clonal expansion. Further using an integrative multi-omics approach combining the transcriptome, TCR repertoire, and open chromatin profile of CD4+ naïve (CD4-TN) and memory subsets, we discovered a stem-cell memory subset that is pre-committed to CTL program within the CD4+ T cell lineage. Through an in vitro differentiation model we developed CD4+ T cells with cytolytic potential from CD4-TN cells. The in vitro differentiated cells followed the trajectory of different developmental memory subsets from ex vivo CD4+ T cells for transcriptomic patterns as well as open-chromatin landscape. Thus, through this model, we deciphered the molecular signatures of early commitment of CD4-TN cells to cytotoxicity program. Of particular interest was the expression of both longevity as well as cytotoxicity associated gene sets by the in vitro differentiated CD4-CTLs, hence generating long-lived CD4-CTL effectors. This specific property can be further explored for vaccine development as well as testing the efficacy and cell based therapies for precision medicine. Overall design: PBMCs were isolated from healthy human blood bank donors (n=8), and pure population of interest was sorted using FACS. scATAC-Seq was performed on these sorted cells after pooling cells from all donors for equal representation across donors.

表达颗粒酶、穿孔素等溶细胞分子的CD4阳性T细胞（CD4+ T cells）已在多种疾病中被检测到，且被证实富集于人类表达CD45RA的效应记忆T细胞亚群（TEMRA），但其起源仍不明确。然而，相较于经典定义的细胞毒性T淋巴细胞（CTLs，即CD8阳性CTLs），这类细胞的基因表达谱尚未得到充分阐释，因此其相关生物学意义仍存在争议。为此，本研究通过并行分析CD4阳性CTLs（CD4-CTLs）与CD8阳性CTLs（CD8-CTLs），证实二者在溶细胞程序上无显著差异，且具有相似的基因表达谱与T细胞抗原受体（TCR）克隆扩增特征。进一步结合转录组、T细胞受体库与开放染色质谱，通过整合多组学方法分析CD4阳性初始T细胞（CD4-TN）及其记忆亚群，我们在CD4+ T细胞谱系中发现了一类预先定向于CTL程序的干细胞样记忆亚群。通过构建体外分化模型，我们从CD4-TN细胞诱导获得了具有溶细胞潜能的CD4+ T细胞。体外分化得到的细胞，其转录组特征与开放染色质景观均遵循离体CD4+ T细胞的不同发育性记忆亚群的分化轨迹。借助该模型，我们解析了CD4-TN细胞向溶细胞程序早期定向的分子特征。尤为值得关注的是，体外分化获得的CD4-CTLs同时表达长寿相关与细胞毒性相关基因集，因此可产生长寿的CD4-CTL效应细胞。这一独特特性可进一步应用于疫苗开发，以及精准医学领域细胞治疗的疗效评估。整体实验设计：从8名健康血库供者的血液中分离外周血单个核细胞（PBMCs），通过荧光激活细胞分选术（FACS）纯化目标细胞群；将所有供者的分选细胞混合以保证各供者的细胞占比均衡，随后对混合细胞进行单细胞ATAC测序（scATAC-Seq）。