Scherle PA, Palladino G, Gerhard W. site, important for targeting local protective responses in vaccines and immunotherapies. immunity is due to T cells resident in the tissue versus those rapidly recruited from other sites. Defining the protective T cell subsets for specific compartments is critical to the successful design of vaccines which target tissue sites. Respiratory infection with influenza virus results in the generation and dissemination of memory T cells into lung and lymphoid tissue sites Aminocaproic acid (Amicar) as demonstrated in mouse models (4C5), and in human Rabbit Polyclonal to ATPBD3 peripheral blood and lungs (6C7). We recently showed that heterogeneous populations of influenza-specific memory CD4 T cells in mice could direct rapid lung viral clearance independent of CD8 T cells and B cells, although did not protect from morbidity of infection (9C10). As circulating memory CD4 T cells reactive to pandemic influenza strains have been identified in healthy humans (8), further insights into the nature of protective memory CD4 T cells could be beneficial in optimizing this potent response. In this study, we identify a population of influenza specific memory CD4 T cells in lung which are noncirculating and exhibit lung-tropic migration independent of antigen-specificity. We used parabiosis models to demonstrate irreversible retention of lung-tropic memory CD4 T cells in the lung, and labeling to identify a non-circulating lung resident polyclonal memory CD4 T cell population persisting after influenza infection–both exhibiting elevated CD69 and CD11a expression. Mice with memory CD4 T cells exclusively in the lung were protected from morbidity and mortality of influenza infection, with spleen-derived memory CD4 T cells affording little protection and even exacerbating mortality Aminocaproic acid (Amicar) despite their diverse tissue residence and migration to the lung. Our results define a new class of memory CD4 T cells resident in lung tissue and reveal tissue compartmentalization as a major determining factor for protection in a key mucosal site, with important implications for targeting site-specific immunity in vaccines and immunotherapies. MATERIALS AND METHODS Mice BALB/c mice (8C16 weeks of age) (NCI Biological Testing Branch), RAG2?/? (BALB/c) mice (Taconic Farms, Germantown, NY), and Influenza Hemagglutinin (HA)-TCR transgenic mice (11) on BALB/c (Thy1.2) or BALB/c(Thy1.1) backgrounds were maintained under specific pathogen-free conditions at the University of Maryland, Columbia University, and the University of Connecticut. Protocols were approved by the Institutional Animal Care and use Committees of all institutions. Reagents Fluorescently-conjugated antibodies specific for CD4, CD8, CD44, CD62L, CD69, CD90.1, CD90.2, and CD11a were purchased from BD Pharmingen (San Diego, CA). Influenza HA peptide (110C120, SFERFEIFPKE) Aminocaproic acid (Amicar) was synthesized by the Biopolymer Laboratory (University of Maryland, Baltimore). Aminocaproic acid (Amicar) Influenza virus infection Aminocaproic acid (Amicar) Mice were infected intransally with 100C500 TCID50 Influenza virus (A/PR/8/34) for sublethal infection and 5000 TCID50 PR8 Influenza (2LD50) for lethal infection as described (9C10, 12). Morbidity was monitored by daily weighing and examination. Influenza virus titers were determined by Tissue Culture Infectious Dose 50 assay (TCID50) as described (13). Generation of influenza-specific memory CD4 T-cells HA-specific memory CD4 T-cells were generated by adoptive transfer of homing assays, HA-specific memory CD4 T cells (Thy1.1+) were isolated from spleens and lungs of RAG2?/? hosts, transferred (2106/mouse) into BALB/c recipient mice, and recipient tissues were recovered 7 and 21 days later. For parabiosis experiments, BALB/c recipients of 2106 lung or spleen HA-specific memory CD4 T cells were surgically conjoined seven days post-transfer to BALB/c mouse partners as described (16), and tissues were harvested from mouse pairs 8C21 days later. antibody labeling and flow cytometry For antibody labeling,.