Single-cell multiomics of CD4+ T cell clones in HIV-1 infected individuals

The clonal expansion of HIV-1-infected CD4+ T cells is a major barrier to cure. Using single-cell ECCITE-seq, we examined the transcriptional landscape, upstream immune regulators, HIV-1 RNA expression, and T cell clonal expansion dynamics of 215,458 CD4+ T cells (267 HIV-1 RNA+ cells and 68 expanded HIV-1 RNA+ T cell clones) from six HIV-1-infected individuals (during viremia and after suppressive antiretroviral therapy) and two uninfected individuals, in unstimulated conditions and after CMV and HIV-1 antigen stimulation. We found that despite antiretroviral therapy, antigen and TNF responses persisted and shaped T cell clonal expansion. HIV-1 resided in Th1 polarized, antigen-responding T cells expressing Bcl-2 family anti-apoptotic genes. HIV-1 RNA+ T cell clones were larger in clone size, established during viremia, persistent after viral suppression, and enriched in GZMB+ cytotoxic effector memory Th1 cells. Targeting HIV-1-infected cytotoxic CD4+ T cells and drivers of clonal expansion provides a new direction for HIV-1 eradication. Study was completed in two arms, unstimulated and antigen stimulated CD4+ T cells. Paired PBMC samples from 6 individuals from the Sabes cohort during acute/recent viremia were used in both arms, 2 uninfected controls were recruited for comparison. For the unstimulated arm, CD4+ T cells were isolated from PBMC by negative selection and stained with a CITE-seq panel. For the stimulated arm, CD8+ T cells were depleted from PBMC. CD8- PBMC were pulsed with either CMV peptides or HIV peptides for 9 hours in the presence of T20 ART. Cells were then stained and antigen specific cells sorted on a FACS ARIA sorter for CD3+CD8-CD19-CD56- Acitvation inducible Marker (AIM, CD69+CD154+). Memory cells were also sorted as CD3+CD8-CD19-CD56-CD69-CD154-CD45RO+. Sorted cells were then hashed and stained with the CITE panel as previous. In either case, labeled cells were loaded into the 10X chromium controler for 5' immune profiling with feature barcoding. This resulted in libraries for the single cell transcriptome, surface proteome, and T cell receptor sequences. Libraries were sequenced with either the minimum requirements (R1 26, R2 90 for transcriptome and CITE-seq) on a HiSeq 4000 or in 150PE mode on a Novaseq 6000. In parallel, bulk TCR repetiore libraries were generated from remaining unstimulated CD4+ T cells using the NEBNext Human ImmuneProfiling kit and sequence on a MiSeq in 300PE mode.

HIV-1感染的CD4+ T细胞克隆扩增是实现艾滋病治愈的主要障碍。本研究借助单细胞ECCITE-seq（single-cell ECCITE-seq）技术，对来自6名HIV-1感染者（分别处于病毒血症期及接受抑制性抗反转录病毒治疗后）与2名未感染者的共计215458个CD4+ T细胞（含267个HIV-1 RNA阳性细胞及68个扩增的HIV-1 RNA阳性T细胞克隆）开展了系统性分析，覆盖转录组图谱、上游免疫调控因子、HIV-1 RNA表达水平以及T细胞克隆扩增动态特征；实验设置了未刺激条件、巨细胞病毒（Cytomegalovirus, CMV）抗原刺激及HIV-1抗原刺激三种处理环境。
研究发现，即便接受抗反转录病毒治疗，抗原与肿瘤坏死因子（Tumor Necrosis Factor, TNF）应答仍持续存在并调控T细胞克隆扩增。HIV-1定植于表达Bcl-2家族抗凋亡基因的辅助性T细胞1（T helper cell 1, Th1）极化型抗原应答T细胞中。HIV-1 RNA阳性T细胞克隆具有更大的克隆体量，形成于病毒血症期，在病毒抑制后仍可持续存在，且富集于表达颗粒酶B（Granzyme B, GZMB）的细胞毒性效应记忆型Th1细胞中。靶向感染HIV-1的细胞毒性CD4+ T细胞及克隆扩增驱动因子，为HIV-1根除治疗提供了全新方向。
本研究分为两个实验组：未刺激组与抗原刺激组，均针对CD4+ T细胞展开。两组实验均使用来自Sabes队列中6名急性/近期病毒血症期感染者的配对外周血单个核细胞（Peripheral Blood Mononuclear Cell, PBMC）样本，并招募2名未感染个体作为对照用于比对分析。
未刺激组中，研究人员通过阴性筛选法从PBMC中分离CD4+ T细胞，并用CITE-seq（Cellular Indexing of Transcriptomes and Epitopes by Sequencing）抗体组合进行染色标记。抗原刺激组中，研究人员先从PBMC中去除CD8+ T细胞，随后将去除CD8+ T细胞的PBMC分别用CMV肽段或HIV-1肽段脉冲刺激9小时，同时添加T20抗反转录病毒药物。随后对细胞进行染色，并借助FACS ARIA分选仪分选抗原特异性细胞，分选门设置为CD3+CD8-CD19-CD56- 激活诱导标志物（Activation Inducible Marker, AIM, CD69+CD154+）。同时还分选了记忆性T细胞，分选门为CD3+CD8-CD19-CD56-CD69-CD154-CD45RO+。分选得到的细胞随后进行多重条码标记，并按此前方案用CITE-seq抗体组合进行染色。
两种实验组的标记细胞均被加载至10X Genomics Chromium控制器中，借助特征条码标记开展5'端免疫组学分析，最终构建得到单细胞转录组、表面蛋白组及T细胞受体（T Cell Receptor, TCR）序列测序文库。测序文库可在HiSeq 4000测序平台上以最低测序参数（转录组及CITE-seq文库的R1读长26bp、R2读长90bp）进行测序，或在NovaSeq 6000测序平台上以150PE模式完成测序。
与此同时，研究人员利用NEBNext人类免疫组库分析试剂盒，从剩余未刺激的CD4+ T细胞中构建批量TCR免疫组库文库，并在MiSeq测序平台上以300PE模式完成测序。