PROJECT SUMMARY The La Jolla CCHI team will investigate pathogen-specific memory T and B cell responses in the blood, and in lymphoid and non-lymphoid tissues in the upper airways, where many airborne infectious pathogens entry and replicate. In Project 3, we will focus on understanding the factors (smoking status, older age, diabetes, vaccination) associated with the magnitude, persistence and quality of pathogen-specific tissue-resident memory T cells (TRM cells) in the upper airway tissue. Upper airway TRM cells play important roles in protection against infection by airborne pathogens. Despite their importance, very little is known about the factors like vaccination routes, co-morbidities like age, diabetes, smoking status that influence the generation of pathogen/vaccine- specific TRM cells in human upper airway tissue. We have extensive expertise in understanding the biology of TRM cells, having shown their importance in driving anti-PD1 therapy T cell responses in human cancers. Here, we will examine the biology of infection-elicited and vaccine-elicited TRM cells in upper airway tissue samples obtained during routine surgery from 200 living donors. These donors are relatively fit for elective surgery and are enrolled in TARGET head and neck study, a prospective observational study that grows by ~20 cases/month at tertiary health centers, and now includes vaccination (TARGET vaccination) as an intervention prior to surgery. Our studies will expand well beyond SARS-CoV-2 to understand the diversity of TRM cells specific to viral (Influenza, respiratory syncytial virus, parainfluenza, metapneumovirus, varicella zoster virus, human papilloma virus), bacterial (pertussis) and fungal pathogens. Studies in Aim 1 will determine the correlation of three important immunomodulating co-morbidities (smoking, older age and diabetes) with features (frequency, phenotype and persistency) of pathogen/vaccine-specific TRM cells in the upper airway tissue. We will test the hypothesis that key co-morbidities like smoking, older age and diabetes are associated with defects in pathogen- specific TRM cell responses in airways. For studies in Aim 2, we will vaccinate donors 2-4 weeks prior to surgery and determine vaccine-responsive TRM cells in upper airway tissue. We hypothesize that parenteral (i.m.) vaccination can also induce/boost (pre-existing) TRM responses in the upper airways. This hypothesis is based on recent studies in model organisms and humans, which suggests that parental vaccination may induce/boost TRM responses in lungs. Because most donors would have had prior exposure to the antigens present in these vaccines by either infection or vaccination – a real-life scenario – our analysis will largely assess effects on pre- existing memory T cells. Overall, studies proposed in this Project that complement studies in Project 1 & 2 will provide important insights into the nature of TRM responses in humans.