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.