Single-cell atlas of lung tissue-resident memory T cells reactive to upper and lower respiratory tract pathogens

项目总结 La Jolla HIPC团队将专注于导致呼吸道传染病的常见病原体。 在项目3中，我们将确定肺组织驻留记忆T细胞(TRM细胞)的分子特性 它们针对广泛的病毒、细菌和真菌病原体。我们的目标是建立一个单细胞 病原体反应性肺TRM细胞转录组、表观基因组和T细胞受体(TCR)图谱。 在目标1中，我们将对从肺中分离的T细胞进行单细胞RNA-SEQ、ATAC-SEQ和TCR-SEQ分析 100例活体供者的组织和支气管肺泡灌洗(BAL)，这些供者因疑似患者而接受手术切除 早期肺癌(靶肺研究)。我们将利用他们的TCR序列来确定病原体- 肺TRM细胞的特异性。为了实现这一目标，我们将生成一个经过实验验证的TCR目录 针对不同病原体表位和HLA型供体的序列；我们将通过以下方式实现这一目标 用靶向感染肺部的特定病原体的肽库刺激匹配捐赠者的PBMC AS：病毒(流感、呼吸道合胞病毒、副流感、偏肺病毒、SARS-CoV-2、普通感冒 细菌(肺炎球菌、百日咳、分枝杆菌)和真菌(曲霉和念珠菌) 病原体，并检索反应T细胞的TCR序列。纵向评估：评估 病原体特异性肺TRM细胞的持久性和可塑性，在供者亚群(n~25-50)中，我们将获得 初步评估后1年采集BALL样本，并如上所述进行单细胞分析。在目标2中，我们将定义 人肺T细胞对疫苗免疫应答的分子特性(n=100)在100个捐赠者中(目标肺 研究)，我们将评估4种不同疫苗的效果(n=25/疫苗队列)：(I)灭活流感疫苗，(Ii) 肺炎球菌疫苗(PPSV23)，(Iii)SARS-CoV-2疫苗，(Iv)百日咳疫苗(Boostrix-IPV)。我们会 比较循环和肺T细胞(TRM细胞)对靶向疫苗的反应的分子特征 不同的病原体。此外，我们将确定血液中对疫苗有反应的T细胞是否真的反映了TRM 肺组织中的反应。纵向评估：我们还将收集重复的血液和BAL样本， 经研究支气管镜检查获得，接种后12个月。我们将确定震级和 检测血液和肺部对疫苗有反应的T细胞的表型以评估其持久性和可塑性。我们 与项目1和2有广泛的协同效应，特别是专注于生成各种免疫签名 呼吸道病原体特异性T细胞。

PROJECT SUMMARY La Jolla HIPC team will focus on common pathogens causing infectious diseases of the respiratory tract. Here in Project 3, we will determine the molecular properties of lung tissue-resident memory T cells (TRM cells) that are specific to a wide-range of viral, bacterial and fungal pathogens. Our goal is to establish a single-cell atlas of the transcriptome, epigenome and T cell receptor (TCR) repertoire of pathogen-reactive lung TRM cells. In Aim 1, we will perform single-cell RNA-seq, ATAC-seq, and TCR-seq analysis of T cells isolated from lung tissue and bronchoalveolar lavage (BAL) of 100 living donors, who undergo surgical resection for suspected early-stage lung cancer (TARGET Lung study). We will utilize their TCR sequences to determine the pathogen- specificity of lung TRM cells. To achieve this goal, we will generate a catalogue of experimentally-validated TCR sequences that are specific to different pathogen epitopes and HLA-typed donors; we will achieve this goal by stimulating PBMCs of matched donors with peptide pools targeting specific pathogens that infects the lungs such as: viral (Influenza, respiratory syncytial virus, para influenza, meta pneumovirus, SARS-CoV-2, common cold corona virus), bacterial (pneumococcus, pertussis, mycobacterium) and fungal (aspergillus and candida) pathogens, and retrieve TCR sequences of responding T cells. Longitudinal assessments: To assess persistence and plasticity of pathogen-specific lung TRM cells, in a subgroup of donors (n~25-50), we will obtain BAL samples 1 year after initial assessments and perform single-cell analysis as above. In Aim 2, we will define the molecular properties of human lung T cells responding to vaccination (n=100). In 100 donors (TARGET Lung study), we will assess effects of 4 different vaccines (n=25/vaccine cohort): (i) inactivated influenza vaccine, (ii) pneumococcal vaccine (PPSV23), (iii) SARS-CoV-2 vaccine, (iv) Pertussis vaccine (Boostrix-IPV). We will compare the molecular features of circulating and lung T cells (TRM cells) that respond to vaccines targeting different pathogens. In addition, we will determine if vaccine-responding T cells in the blood truly reflect TRM responses in the lung tissue. Longitudinal assessments: We will also collect repeat blood and BAL samples, obtained by research bronchoscopy, 12 months after vaccination. We will determine the magnitude and phenotype of vaccine-responsive T cells in the blood and lungs to assess their persistence and plasticity. We have extensive synergies with Project 1 and 2, especially the focus on generating immune signatures of various respiratory pathogen-specific T cells.