Supplementary Materials Supplemental Materials (PDF) JEM_20170521_sm. transcriptional profiles enriched for T-betCregulated genes. Notably, mouse and human infant T cells exhibited increased T-bet expression after activation, and reduction of T-bet levels in infant mice enhanced lung TRM establishment. Our findings reveal that infant T cells are intrinsically programmed for short-term responses, and targeting Atenolol key regulators could promote long-term, tissue-targeted protection at this crucial life stage. Introduction Infants exhibit increased morbidity and mortality after respiratory Atenolol infections and experience more repeat infections compared with older children and adults, suggesting impaired protective immunity. The worse outcome Mouse monoclonal to Influenza A virus Nucleoprotein for infants in response to infection and their limited or delayed response to vaccines (Siegrist, 2007) have been attributed to the immaturity of immune responses and to T lymphocytes, in particular, which coordinate adaptive immunity (PrabhuDas et al., 2011). Although differences in T cell subset composition and cytokine profile between infant and adult T cells have been described (Lewis et al., 1991; Gibbons et al., 2014; Thome et al., 2016), the basic mechanisms underlying the regulation of infant T cell responses, including their functional differentiation, localization, and maintenance in response to infection remain undefined. There is a critical need for new insights into infant immune responses to both promote protection in response to infection and maximize efficacy of the multiple vaccines administered in early life. Effective clearance of respiratory pathogens is coupled to establishment of lung-localized effector and memory T cells. In adult mouse models, lung-localized Th1 effector cells producing IFN- are important for directing clearance of primary influenza infection (Graham et al., 1993, 1994). We previously showed that populations of CD4+ and CD8+ lung tissueCresident memory T cells (TRM) are generated in response to influenza infection or i.n. administration of live-attenuated influenza vaccine (LAIV) in mice and that these cells mediate rapid, in situ protective responses to secondary viral challenge (Teijaro et al., 2011; Turner et al., 2014; Zens et al., 2016). In humans, influenza-specific CD4+ and CD8+ T cells with TRM phenotypes have been identified within lung tissue (de Bree et al., 2005; Purwar et al., 2011; Turner et al., 2014), and TRM-phenotype cells comprise the majority of memory T cells in diverse human tissues (Sathaliyawala et al., 2013; Thome et al., 2014). The robust protection mediated by TRM in the lungs and their predominance within multiple tissue sites (Masopust et al., 2001; Wakim et al., 2010; Jiang et al., 2012; Shin and Iwasaki, 2012) suggests that TRMs are an important target for promoting antiviral immunity by vaccines and immunotherapies. The generation of tissue-localized T cell responses within the lung or other sites and the extent to which protective T cell memory and TRMs can be established during infancy have not been well studied. In contrast to adults, most peripheral T cells are naive in early life (Thome et al., 2016) and have distinct patterns of homing receptor expression (Grindebacke et al., 2009; Crespo et al., 2012). Neonatal and infant T cells also exhibit differences in cytokine expression and differentiation after in vitro activation or infection, compared with their adult counterparts (Lewis et al., 1986, 1991; Gibbons et al., 2014; Smith et al., 2014). How such differences affect protection and the generation of lasting T cell memory after infection or vaccination is not known. We hypothesized that reduced protection after infection and decreased vaccine responses observed during infancy could be due to impaired tissue localization of effector T cell responses and/or the establishment of persisting TRM. Using an infant mouse model of influenza infection and vaccination, we found that infants mounted robust, primary, lung-localized CD4+ Atenolol and CD8+ T cell responses to virus infection and LAIV. However, these cells were inefficiently maintained long term as TRM. In reciprocal transfers, we observed reduced lung TRM establishment after infection by infant, compared with adult, CD4+ T cells in either adult or infant hosts, suggesting T cellCintrinsic differences, rather than the lung environment mediating the distinct infant immune responses. We found distinct transcriptional profiles for infant, compared with adult, T cells after short-term activation in vitro and during the acute response to infection in the lung in vivo, with enhanced expression of T-bet and T-betCregulated genes in both conditions. Importantly, reducing T-bet expression during infection promoted lung TRM establishment to near-adult levels, indicating that altered differentiation of infant T cells after infection affects TRM formation. These findings provide mechanistic insight into the process of TRM establishment with important implications for promoting vaccine-mediated protection during early life. Results Infants generate robust, lung-localized primary T cell responses to influenza infection We used an infant mouse model to assess tissue-localized.