CX3CR1hi cells were most abundant during the first ~250 days, but diminished over time, while CX3CR1? TMem cells gradually increased in frequency and became the predominant subset after ~8 months. tissues. As CX3CR1int TMem cells present unique phenotypic, homeostatic and migratory properties, we designate this subset peripheral memory (TPM) cells and propose that TPM cells are chiefly responsible for the surveillance of non-lymphoid tissues. Graphical Abstract INTRODUCTION When na?ve CD8+ T cells (TN) encounter an infection, activation by cognate antigen (Ag) causes them to proliferate and to give rise to effector (TEff) cells that eradicate the pathogen. Eventually, most TEff cells are eliminated, but a small fraction persists as long-lived memory (TMem) cells (Williams and Bevan, 2007). Both TEff and TMem cells are composed of distinct subsets (Jameson and Masopust, 2009; Mueller et al., 2013). At the TEff stage, differential expression of KLRG1 (Killer Cell Lectin Like Receptor G1) and CD127 is commonly used Diphenhydramine hcl to identify differentiation states that differ in their propensity to form memory. The two major known TMem populations in blood and spleen are central memory (TCM) and effector memory (TEM) cells, which are Diphenhydramine hcl traditionally defined by differential expression of the lymph node (LN) homing receptors CD62L and CCR7 (Marzo et al., 2005; Sallusto et al., 1999; Wherry et al., 2003). TCM cells have a higher proliferative capacity and are thought to provide superior protection against reinfection than TEM cells, at least in some settings. TEM Diphenhydramine hcl cells, in contrast, are more cytotoxic than TCM cells. Because na?ve (TN) and TCM cells (but not TEM cells) express CCR7 and CD62L, they can home to LNs via high endothelial venules (HEV) and survey LNs for cognate Ag (von Andrian and Mempel, 2003). After a few hours to days, these migratory T cells egress from LNs and return to the blood via the efferent lymphatics and thoracic duct (TD) (Gowans and Knight, 1964). Some TMem cells are also present in afferent lymphatics that drain interstitial fluid from peripheral tissues into LNs (Mackay et al., 1990). Since TEM cells cannot home directly to LNs via HEV, it had been postulated that circulating TEM cells continuously survey non-lymphoid tissues and return to the blood TLR3 via the draining lymph conduits (Sallusto et al., 1999). To date, this widely held idea has not been tested by rigorous experiments. A third TMem subset – tissue resident memory cells (TRM) – was recently identified (Mueller et al., 2013). This tissue-confined, non-migratory TMem population is derived from TEff cells that seed non-lymphoid tissues Diphenhydramine hcl early after infection (Mackay et al., 2013; Masopust et al., 2010; Stary et al., 2015). It has also been suggested that TRM cells may be progeny of TEM cells (Jiang et al., 2012). In contrast, whether TEM and TCM cells have distinct precursors within the TEff population is unclear, and the rules that determine the differentiation of these TMem subsets remain largely elusive. These uncertainties are due, at least in part, to the lack of phenotypic markers that Diphenhydramine hcl can link TEff differentiation states to specific TMem subsets. Consequently, the relationship between TEM, TCM and TRM cells has been a subject of debate (Marzo et al., 2005; Wherry et al., 2003). Aside from the TCM/TEM paradigm, TMem cells have also been sub-divided based on differential expression of phenotypic markers, including CD27 (Hamann et al., 1997), CD127 (Kaech et al., 2003), KLRG1, CD43 (1B11) (Hikono et al., 2007; Joshi et al., 2007; Olson et al., 2013; Sarkar et al., 2008; Voehringer et al., 2001) and, recently, CX3CR1 (Bottcher et al., 2015). For example, KLRG1?CD27+ TMem cells mount more potent recall responses than KLRG1+ TMem cells (Hikono et al., 2007). Similarly, CX3CR1+ TMem cells exhibit robust cytotoxicity, while CX3CR1? TMem cells are largely non-cytotoxic and possess greater proliferative capacity (Bottcher et al., 2015). The present study was prompted by the observation that in response to lymphocytic choriomeningitis virus Armstrong (LCMV) infection, the CX3CR1+ CD8+ T cell subset could be further subdivided into two distinct populations that express CX3CR1 at intermediate or high levels. Thus, we investigated the properties of CX3CR1?, CX3CR1int and CX3CR1hi TEff and TMem cells and their relationship to the classical TCM, TEM and TRM subsets that arise in response to systemic infections. We demonstrate that CX3CR1int TMem cells represent a distinct subset that differs from TCM (CX3CR1?), TEM (CX3CR1hi) and TRM (CX3CR1?/low) cells in its phenotypic, migratory and homeostatic properties. CX3CR1int TMem cells possessed the highest steady-state self-renewal capacity of all TMem subsets, and were the predominant TMem subset surveying peripheral tissues. RESULTS Viral infection induces CX3CR1 on virus-specific CD8+ TEff cells To monitor CX3CR1 expression during viral infection, LCMV was injected intravenously (i.v.) into locus (Jung et al.,.