Single-cell TCR profiling of CSF and blood cells of PWH reveals persistent and shared infected T cell clones across tissue compartments even after ART

The brain is a site of persistent infected cells during HIV infection. However, the dynamics of central nervous system (CNS) infection and T cell trafficking in people living with HIV (PWH) are incompletely understood. We profiled the single cell T cell repertoire and host transcriptome from paired blood and cerebrospinal fluid (CSF) from PWH and uninfected controls. We detected HIV RNA-producing cells in 8/11 (72.7 %) CSF samples and 6/11 (54.5 %) blood samples, with a higher frequency of infected single CD4 T cells in CSF than in blood. Infected CSF T cells displayed a unique transcriptional profile compared to uninfected CSF T cells. Most infected T cell clones were not shared across tissue; however, in a subset of participants we detected identical T cell clones that were infected in both CSF and blood. Longitudinally following one PWH before and at three time points after initiating ART, we found infected T cell clones that persisted after ART initiation, in both CSF and blood, including a T cell clone that expanded in the CSF several months after ART initiation. Our findings suggest that infected T cells traffic to the CNS where they undergo clonal expansion despite ART, contributing to the CNS reservoir. Asymptomatic PWH P2 - P7 were enrolled (n = 7), and all were stable on ART (median 16 years) with suppressed plasma viral loads (< 50 copies/mL) and a median CD4 count of 503. One additional PWH was enrolled (P1) with newly diagnosed HIV and not yet on ART, and had a plasma viral load of 257,000 copies/mL. Control participants C1 - C6 were healthy volunteers recruited from the surrounding community for research sampling (n = 6) plus one hospitalized individual C1 undergoing a workup for gait instability. All control participants were confirmed HIV-negative by plasma enzyme immunoassay and screened for confounding neurological conditions. All participants consented to large-volume lumbar puncture (up to 30cc CSF removed) and blood draw for research purposes, or to donate additional CSF and blood samples collected during clinical standard-of-care procedures. Participant P1 underwent longitudinal study visits and consented to provide blood and CSF samples at HIV diagnosis/baseline and three, six, and nine months after initiating ART. The gene expression and T cell receptors of the peripheral blood and CSF samples were then measured using 5’ V(D)J 10x Genomics paired scRNA-seq and TCR-seq. Sequencing data for C1 - C3 was previously published on SRA (study accession: SRP312293, C1_BLD_RNA: SRR14076861, C1_BLD_TCR: SRR14076833, C1_CSF_RNA: SRR14076871, C1_CSF_TCR: SRR14076842, C2_BLD_RNA: SRR14076860, C2_BLD_TCR: SRR14076832, C2_CSF_RNA: SRR14076869, C2_CSF_TCR: SRR14076841, C3_BLD_RNA: SRR14076858, C3_BLD_TCR: SRR14076831, C3_CSF_RNA: SRR14076868, C3_CSF_TCR: SRR14076840).

人类免疫缺陷病毒（HIV）感染期间，大脑是持续存在感染细胞的场所。然而，人类免疫缺陷病毒感染者（People Living with HIV, PWH）的中枢神经系统（Central Nervous System, CNS）感染动力学及T细胞迁移机制尚未完全阐明。本研究对HIV感染者与未感染对照者的配对血液及脑脊液（Cerebrospinal Fluid, CSF）样本开展了单细胞T细胞受体库及宿主转录组谱分析。我们在11份脑脊液样本中的8份（72.7%）、11份血液样本中的6份（54.5%）中检测到了表达HIV RNA的感染细胞，且脑脊液中感染的CD4阳性T细胞单克隆频率高于血液。与未感染的脑脊液T细胞相比，感染的脑脊液T细胞呈现独特的转录谱特征。大多数感染性T细胞克隆仅局限于单一组织；然而，在部分受试者中，我们同时在脑脊液与血液中检测到了完全一致的感染性T细胞克隆。我们对1名HIV感染者在启动抗逆转录病毒治疗（Antiretroviral Therapy, ART）前及治疗后的三个时间点进行了纵向随访，结果在脑脊液与血液中均检测到了启动ART后仍持续存在的感染性T细胞克隆，其中1个T细胞克隆在ART启动数月后于脑脊液中发生扩增。本研究结果提示，感染性T细胞可迁移至中枢神经系统，即便在ART治疗期间仍可发生克隆扩增，进而构成中枢神经系统病毒储存库。本研究纳入7名无症状HIV感染者（P2-P7），均接受ART治疗且病情稳定（中位治疗时长16年），血浆病毒载量被抑制至<50拷贝/mL，中位CD4细胞计数为503。另有1名新发诊断HIV感染且尚未启动ART的受试者（P1），其血浆病毒载量为257000拷贝/mL。对照受试者共6名（C1-C6），其中5名为招募自周边社区的健康研究志愿者，另1名C1为因步态不稳接受临床评估的住院患者。所有对照受试者均经血浆酶联免疫吸附试验确认HIV阴性，并排除了混杂性神经系统疾病。所有受试者均同意为研究目的采集大容量腰椎穿刺脑脊液（最多可采集30mL）及血液样本，或同意使用临床常规诊疗过程中剩余的脑脊液与血液样本用于研究。受试者P1接受了纵向随访访视，同意在HIV诊断/基线时以及启动ART后3、6、9个月采集血液与脑脊液样本。本研究通过5’ V(D)J 10x Genomics 配对单细胞RNA测序（single cell RNA-seq, scRNA-seq）与T细胞受体测序（T cell receptor-seq, TCR-seq），对受试者外周血与脑脊液样本的基因表达及T细胞受体特征进行了检测。C1-C3的测序数据此前已发表于序列读取档案库（Sequence Read Archive, SRA），研究编号为SRP312293，相关样本编号如下：C1_BLD_RNA: SRR14076861, C1_BLD_TCR: SRR14076833, C1_CSF_RNA: SRR14076871, C1_CSF_TCR: SRR14076842, C2_BLD_RNA: SRR14076860, C2_BLD_TCR: SRR14076832, C2_CSF_RNA: SRR14076869, C2_CSF_TCR: SRR14076841, C3_BLD_RNA: SRR14076858, C3_BLD_TCR: SRR14076831, C3_CSF_RNA: SRR14076868, C3_CSF_TCR: SRR14076840。