This R01 application proposes to continue work funded for the past five years by a Transformative R01 (?Vaccination to generate tissue resident memory T cells?) from the Office of the NIH Director (TR01?s cannot be renewed as such). The previous funding period allowed Drs. Kupper and Clark to make fundamental insights into immunobiology of TRM in skin and other tissues, in both mouse models and humans. Taken together, the body of work published by both co-PI?s over the past five years validates the importance of TRM in protective immunity, is summarized in commissioned reviews published in 2015 by Dr. Clark (in Science Translational Medicine) and Dr. Kupper (in Nature Medicine). The present application builds on exciting preliminary data showing that tissue specific imprinting occurs very early after T cell activation, and that the gene expression profile of T cells activated in skin draining and lung draining lymph nodes (LN) is surprisingly similar (but very different from gut draining LN). This may explain why immunization with a replication deficient poxvirus (MVA) vector through epidermal disruption (formerly ?skin scarification?, or ed/ss) generates not only skin TRM but also lung TRM with very high efficiency. This suggests that ed/ss skin immunization may be a highly effective way of generating protective immunity in the lung to pulmonary pathogens like influenza. This hypothesis will be tested in Aim 1. Conventional vaccination routes (intradermal, subcutaneous, and intramuscular) were much less efficient at generating TRM in both skin and lung. Remarkably, the gene expression profile of T cells responding to the same antigen delivered by these different routes was very different, with ep/ss and i.m. being the most different. These differences in gene expression were maintained even 30 days later in splenic TEM, and were manifested by a strikingly different capacity to generate TRM. Some of the greatest differences in gene expression between TRM and TCM are in genes relevant to lipid metabolism (fatty acid binding proteins/FABP, lipases, and CD36/LDL receptor); TRM?s generated from T cells deficient in FABP4 and 5 did not persist in skin. The hypothesis that the ep/ss mode of immunization most efficiently generates TRM that are dependent on fatty acid metabolism for survival in peripheral tissues will be tested in Aim 2. Finally, in Aim 3, related hypotheses will be tested in translational studies using human tissue, with blood, skin, and lung tissue being obtained from thoracic surgery patients undergoing lobectomies. We predict, and will test for, overlap (at the level of CDR3 sequence) in the T cell repertoire of skin and lung by high throughput sequencing of the TCRB gene, and will directly compare human skin and lung TRM by gene expression, cell surface phenotype, and function. The previously unappreciated commonalities in skin and lung homing T cells may explain phenomena from the tuberculin skin test to the ?atopic march?, where atopic dermatitis is followed by asthma, and has important implications for human vaccine development against pathogens that enter the host through lung or skin.