The large number of directly conjugated antibodies available for flow cytometry should make this technique readily adaptable to study the major immune cell types present in alveolar tissues. CD103/CD20 antibodies directly into the lung and were able to detect TRM/BRMfollowingin situlabelling within seconds of directintra-alveolardelivery of microdoses of fluorescently labelled antibodies. == Discussion == In situ, no wash, immunolabelling withintra-alveolarOEM imaging is a novel methodology with the potential to expand the experimental utility of EVLV and pre-clinical models. Keywords:resident memory T cells, resident memory B cells, lung, optical endomicroscopy, fluorescence lifetime imaging == Introduction == Human and non-human primate (NHP) studies have highlighted the protective role of lung-resident memory T cells (TRM) in controlling respiratory pathogens (13). Constituting the majority of T cells within the lung (4), TRMhave a unique phenotype that differentiates them from other memory T cell subsets and their sequestration within the lung offers an ideal location to respond to respiratory infections (5). Requiring only cognate antigen for their activation (6), TRMare Ednra highly proliferative, producing polyfunctional progeny with superior effector function (7,8). Activated CD4+and CD8+TRMin the lungs of patients withMycobacterium tuberculosis(MTB) limit intracellular MTB replication in macrophages (9), whilst increased numbers of CD8+TRMin BAL is associated with reduced symptoms and viral load following human respiratory syncytial BI01383298 virus (RSV) infection (1). In BI01383298 human influenza infection, CD4+and CD8+TRMconfer heterosubtypic protection (10), with CD8+TRMrecognising universally conserved peptides expressed by influenza A, B and C viruses (11). Virus-specific pulmonary CD8+TRMalso have shown innate like properties that amplify inflammation and enhance neutrophil recruitment BI01383298 following noncognate bacterial infection to aid clearance, as demonstrated in murine models (12). While most actions of TRMbenefit the host, following chronic exposure to allergens, TRMcan also be detrimental and contribute significantly to pathology in experimental allergic asthma (13,14), indicating monitoring the frequency and phenotype of TRMhas relevance beyond respiratory infections. Antigen-experienced lungs are also enriched with B cells expressing a resident memory phenotype (15). Murine parabiosis studies have demonstrated that pulmonary-resident memory B cells (BRM) require local antigen encounter and early CD40/CD40 ligand interactions with T cells for their formation and are phenotypically and functionally distinct from their systemic counterparts (5,16). Murine BRMplay a protective role in response to both viral influenza (1618) and bacterial pneumococcal pneumonia (15) infections, providing rapid antibody secreting cells (ASC) capable of producing a range of class switched, cross-reactive antibodies to confer heterosubtypic protection (1517). Vaccination strategies designed to induce pulmonary resident memory lymphocyte populations have been successfully demonstrated in murine models (19). In NHP, pulmonary mucosal delivery of MTB vaccine is associated with enhanced protection over standard intradermal immunizationviathe induction of lung TRM(20). As TRMand BRMformation is dependent on pulmonary cognate antigen encounter (16,21), its anticipated that pulmonary mucosal vaccination will also generate BRM. This can induce higher levels of local immunity than that generated by natural MTB infection (20). Strategies that establish antigen-specific TRMand BRMpopulations or enhance pre-existing populations have the potential to provide local long lasting heterosubtypic protection where it is most needed. Current methods for evaluating respiratory vaccine efficacy, however, rely mostly on peripheral blood sampling to reveal the level of humoral protection and circulating central memory T cell (TCM) and effector memory T cell (TEM) populations. However, as TRMand BRMdo not circulate and limited blood biomarker surrogates exist, current methods for assessing local immunity are limited. These studies highlight the pivotal role of TRMand BRMin orchestrating and directing protective responses to respiratory infections. The spatial and temporal characterisation of such pulmonary resident immune cells is thus of key importance, but most available methods require biopsy, tissue digestion and post-mortem studies (16,22). In this study, we aimed to develop anin situmethod for the direct detection of these cells in the human lung by exploiting recent advances inin situ-optical endomicroscopy, within the BI01383298 alveoli (23), which may provide a valuable tool for quantifying pulmonary resident memory populations following infection and vaccination. The objective was to develop a technical platform for direct alveolar imaging of pulmonary TRMand BRM.We aimed to develop anin situimmunolabelling approach with a clinic-ready fibre-based optical endomicroscopy (OEM) system that combined both intensity imaging and fluorescence lifetime imaging (FLIM) to ensure contrast and specificity was achieved over endogenous elastin fluorescence of the lung. With NHP providing an ideal model for translational studies, this project also aimed to establish a cross-reactive antibody panel capable of detecting TRMand BRMin both humans and NHP species. FLIM is a fluorescence imaging technique where the contrast is based on the lifetime of individual.