Sifting through CD8+ T Cell Memory

Immunity

Previews Sifting through CD8+ T Cell Memory Matthew D. Martin1 and Vladimir P. Badovinac1,2,* 1Department

of Pathology Graduate Program in Immunology University of Iowa, Iowa City, IA 52242, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.immuni.2016.12.005 2Interdisciplinary

In this issue of Immunity, Gerlach et al. (2016) describe three distinct memory CD8+ T cell subsets based upon expression of the fractalkine receptor CX3CR1. Their findings revise the paradigm of effector and central memory T cells by revealing a subset of CD8+ memory T cells defined by intermediate levels of expression of CX3CR1 that conducts tissue surveillance. Upon infection, pathogen-specific naive CD8+ T cells proliferate and differentiate into a heterogeneous pool of effector cells. While most cells die during contraction, a fraction remains to form the longlived memory CD8+ T cell pool. Because memory CD8+ T cells provide protection against re-infection, researchers have sought to identify factors influencing survival of effector cells that populate the memory pool. Differential expression of surface proteins has been used to divide heterogenous memory CD8+ T cells into subsets with differing functional abilities, and circulating memory CD8+ T cells have classically been divided into effector (Tem) and central (Tcm) memory cells. Tcm cells express CCR7 and CD62L, which allows them to home to the lymph nodes (LN) where they survey for cognate antigen (Ag) (Sallusto et al., 1999; von Andrian and Mempel, 2003). In addition, they are better equipped to produce interleukin-2 (IL-2), to persist within the host, and to proliferate following systemic infections (Wherry et al., 2003). Conversely, Tem cells display a higher degree of cytotoxicity and have been thought to play the role of tissue surveyor. As an additional layer of complexity in defining the memory CD8+ T cell pool, the phenotype and function of cells comprising the memory population continues to change with time following infection (Eberlein et al., 2016; Martin et al., 2015), and Tcm cells become the predominant memory CD8+ T cell subset with time. While the Tem /Tcm paradigm has allowed for the identification of memory CD8+ T cells of differing functional capabilities and informed our thinking on the lineage relationships of memory subsets, the roles of distinct CD8+ T cells and the rules governing dif-

ferentiation of diverse memory CD8+ T cells remains incompletely defined. For this reason, others have used strategies to classify memory CD8+ T cells outside of the Tem/Tcm paradigm based upon expression of markers including CD43 (1B11) (Hikono et al., 2007), and more recently based upon expression of CX3CR1 (Bo¨ttcher et al., 2015). In this issue of Immunity, von Andrian and colleagues use expression of CX3CR1 to define three distinct effector and memory CD8+ T cell subsets (Gerlach et al., 2016). They use this subsetting strategy to show that expression of CX3CR1 predicts effector cells that differ in their propensity to generate long-lived memory and to sift through the memory CD8+ T cell pool to identify subsets with unique attributes. In agreement with a recent report (Bo¨ttcher et al., 2015), Gerlach et al. (2016) found that infection of Cx3cr1+/gfp reporter mice with LCMV or vaccinia virus led to an increase in the percentage of Ag-specific CD8+ T cells expressing CX3CR1. However, whereas Bo¨ttcher et al. (2015) identified cells as either CX3CR1 positive or negative, Gerlach et al. (2016) identified three effector cell populations consisting of CX3CR1hi, CX3CR1int, and CX3CR1 CD8+ T cells. Interestingly, adoptive transfer of sorted CX3CR1hi, CX3CR1int, and CX3CR1 CD8+ T cells into infection-matched mice revealed that CX3CR1 cells are able to generate the greatest number of progeny. CX3CR1hi cells produced primarily CX3CR1hi cells, CX3CR1int cells produced a mix of mostly CX3CR1hi and CX3CR1int cells, and CX3CR1 cells produced roughly equal proportions of all three subsets. Thus, CX3CR1 expression can be used to distinguish effector CD8+

1184 Immunity 45, December 20, 2016 ª 2016 Elsevier Inc.

T cells of differing capacities to generate memory cells and to predict the phenotype of memory progeny. The authors next examined whether CX3CR1 expression identifies memory subsets of distinct function, and the relationship of CX3CR1hi, CX3CR1int, and CX3CR1 subsets to classical Tem and Tcm subsets. All three subsets persisted following the effector-to-memory transition, and expression of CX3CR1 identified three distinct memory CD8+ T cell subsets with differing functionality. The function of CX3CR1hi cells overlapped with classically defined Tem cells and CX3CR1 cells shared attributes of Tcm cells. Interestingly, while CX3CR1int cells were able to proliferate to a similar extent as CX3CR1 cells during recall responses, functional abilities including IL-2 production, cytotoxicity, and LN homing of CX3CR1int cells were intermediate between Tem and Tcm cells, and only approximately half of CX3CR1int cells became CD62L+ 1 year after infection. Thus, subsetting memory CD8+ T cells based upon CX3CR1 expression might reduce heterogeneity within Tem and Tcm subsets based upon current identification strategies and allow bona fide Tem and Tcm cells to be sifted from the memory population. At present, the lineage relationships of Tem and Tcm subsets is unclear. Compelling arguments have been made that either conversion to the Tcm subset occurs in a linear fashion (Tem-to-Tcm conversion) (Wherry et al., 2003), or, in stark contrast, that Tem and Tcm cells comprise distinct lineages (Marzo et al., 2005). The much-needed clarification came when the authors sorted CD62Lsubsets of memory CD8+ T cells based

Immunity

Previews

Figure 1. Tissue Surveillance for memory CD8+ T Cell Subsets Effector memory CD8+ T cells (Tem), defined by high expression of CX3CR1 and low expression of CD62L (CD62Ll0CX3CR1hi), primarily circulate within the blood and spleen (intravascular – ivpos) and are excluded from the extravascular areas (ivneg) of lymphoid and non-lymphoid tissues, indicating that they do not play a role in tissue surveillance. Central memory CD8+ T cells (Tcm; CX3CR1 ) cells can be found within the extravascular areas of lymphoid and non-lymphoid tissues, they primarily circulate within lymph fluid and localize to secondary lymphoid organs where they survey for cognate antigen (Ag) due to their high expression of CD62L and CCR7. Tissue surveillance is carried out by peripheral memory CD8+ T cells (Tpm; CX3CR1int), which are found in the extravascular areas of lymphoid and non-lymphoid tissues and are highly represented in the lymphatics draining peripheral tissues. T resident memory (Trm) cells do not re-circulate, but rather reside within peripheral tissues where they can sense and respond to localized infections. The observation that CX3CR1hi cells are absent in peripheral tissues suggests that Trm cells are seeded from the CX3CR1 and/or CX3CR1int populations, and CX3CR1 expression-based analysis might provide insight into the lineage of true Trm cells.

on the expression of CX3CR1 and transfered them into new hosts to follow their conversion to CD62L+ Tcm cell phenotype. A portion of sorted CD62L CX3CR1 and CX3CR1int cells acquired expression of CD62L while CD62L CX3CR1hi cells remained CD62L . Thus, bona fide Tem cells are unable to convert to Tcm cells while a proportion of CD62L cells that are CX3CR1 and CX3CR1int are able to acquire CD62L expression. Because Tem cells cannot localize to the LNs via high endothelial venules, they have been thought to play the role of tissue surveyor. Interestingly, intravascular staining revealed that CX3CR1hi cells were largely absent from tissues, calling into question the proposed role of Tem cells as tissue surveyors. CX3CR1int cells, on the other hand, were the most abundant subset in thoracic duct lymph (TDL), suggesting that this subset might be primarily responsible for tissue surveillance. Indeed, a series of elegant parabiosis experiments utilizing wild-type mice, lymphotoxin-a-deficient mice, and parabionts treated with anti-CD62L led to the surprising conclusion that the CX3CR1int subset rather than Tem cells survey peripheral tissues, and that they preferentially recirculate through lymph nodes via a CD62L independent route. Because of this role of CX3CR1int cells in tissue surveillance, the authors designate the subset as ‘‘peripheral memory’’ (Tpm) cells.

Overall, by subsetting effector and memory CD8+ T cells into CX3CR1 , CX3CR1int, and CX3CR1hi populations, Gerlach et al. (2016) provide an additional means of identifying cells of distinct function that will spur further experimentation designed to better understand immune surveillance by unique CD8+ T cell populations. The results indicate that CX3CR1 can be used as a marker to identify effector CD8+ T cells that differ in the ability to form long-lived memory, and expression of CX3CR1 in combination with other markers might allow for a more precise identification of Tem and Tcm subsets and serve as a way to probe relationships between Tem, Tcm, and other memory T cell subsets including bona fide tissueresident memory cells (Trm). Many of the phenotypic and functional properties that have been ascribed to Tem and Tcm cells are reinforced in this study. However, tissue surveillance as defined by the ability to traverse the lymphatics entering and draining peripheral tissues, a role previously attributed to Tem cells, is primarily conducted by Tpm cells (Figure 1). This study opens a number of interesting avenues for researchers to pursue. For instance, half of Tpm cells acquire CD62L expression one year after infection and representation of Tpm cells is reduced but not eliminated in the absence of Tbet. Does this suggested heterogeneity inside the Tpm subset bear any significance, and can it potentially be used to

fine tune our understanding of division of labor within the Tpm subset and memory in general? The signals that regulate CX3CR1 expression and promote survival of subsets expressing differing levels of CX3CR1 are relatively ill-defined. The authors suggest that yet-to-be-defined signals that promote CX3CR1 expression might be restricted to the SLO or the circulatory system as CX3CR1hi cells are absent from non-lymphoid tissues. Whether superior basal turnover of Tpm cells compared to Tcm cells as observed by the authors is purely explained by their broader migratory pattern and better chances to encounter survival signals (ex. IL-15) despite similar expression of CD122 or if other mechanism(s) control the observed phenomenon has to be experimentally addressed. In addition, the absence of CX3CR1hi cells in peripheral tissues also suggests that Trm cells are seeded from the CX3CR1 and/or CX3CR1int populations. Thus, CX3CR1 expression-based analysis might provide insight into the lineage of true Trm cells. Finally, (Gerlach et al., 2016) suggest that tissue surveillance is conducted by CX3CR1int Tpm cells rather than Tem cells, which necessitates a change in thinking about the roles that different CD8+ T cell subsets play in immunosurveillance and, ultimately, in protection against re-infection. Protective capacity of memory CD8+ T cell subsets depends on the nature of the infection (Nolz and Harty, 2011) and this one-size-does-not-fit-all concept has to be included in rational CD8+ T cellbased vaccine design. Therefore, a critical step moving forward will be to specifically define the role of Tcm, Tem, and Tpm subsets in providing protection to a wide variety of pathogens. In parallel, deciphering the precise mechanisms that govern generation and maintenance of all three subsets could aid in the development of vaccines designed to elicit large numbers of memory CD8+ T cells of a phenotype and/or function best suited to provide protection against specific pathogens. REFERENCES Bo¨ttcher, J.P., Beyer, M., Meissner, F., Abdullah, Z., Sander, J., Ho¨chst, B., Eickhoff, S., Rieckmann, J.C., Russo, C., Bauer, T., et al. (2015). Nat. Commun. 6, 8306. Eberlein, J., Davenport, B., Nguyen, T., Victorino, F., Haist, K., Jhun, K., Karimpour-Fard, A., Hunter,

Immunity 45, December 20, 2016 1185

Immunity

Previews L., Kedl, R., Clambey, E.T., and Homann, D. (2016). J. Clin. Invest. 126, 3942–3960. Gerlach, C., Moseman, E.A., Loughhead, S.M., Alvarez, D., Zwijnenbur, A.J., Waanders, L., Garg, R., de la Torre, J.C., and von Andrian, U.H. (2016). Immunity 45, this issue, 1270–1284. Hikono, H., Kohlmeier, J.E., Takamura, S., Wittmer, S.T., Roberts, A.D., and Woodland, D.L. (2007). J. Exp. Med. 204, 1625–1636.

Martin, M.D., Kim, M.T., Shan, Q., Sompallae, R., Xue, H.H., Harty, J.T., and Badovinac, V.P. (2015). PLoS Pathog. 11, e1005219. Marzo, A.L., Klonowski, K.D., Le Bon, A., Borrow, P., Tough, D.F., and Lefranc¸ois, L. (2005). Nat. Immunol. 6, 793–799. Nolz, J.C., and Harty, J.T. (2011). Immunity 34, 781–793.

Sallusto, F., Lenig, D., Fo¨rster, R., Lipp, M., and Lanzavecchia, A. (1999). Nature 401, 708–712. von Andrian, U.H., and Mempel, T.R. (2003). Nat. Rev. Immunol. 3, 867–878. €ber, V., Becker, T.C., MasoWherry, E.J., Teichgra pust, D., Kaech, S.M., Antia, R., von Andrian, U.H., and Ahmed, R. (2003). Nat. Immunol. 4, 225–234.

Immune Cell Intolerance for Excess Cholesterol Scott B. Widenmaier1 and Go¨khan S. Hotamıs¸lıgil1,2,* Genetics and Complex Diseases and Sabri U¨lker Center, Harvard T.H. Chan School of Public Health, Boston, MA 02115, USA Institute of Harvard and MIT, Cambridge, MA 02142, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.immuni.2016.12.006 1Department of 2Broad

Chronic metabolic challenges have severe consequences on physiological systems. In this issue of Immunity, Ito et al. (2016) show that defects in cholesterol metabolism in CD11c+ immune cells result in impaired antigen presentation and ultimately in autoimmune disease. Chronic metabolic disease, in all its forms, has emerged as one of the most significant threats to human health in the 21st century. This is in part due to the detrimental impact of metabolic stress on critical physiological processes such as hepatic and renal clearance of toxins, cardiac output, respiration, and cerebral blood flow. A growing body of experimental and clinical evidence supports the notion that metabolic states and dietary habits impact immune system homeostasis and function. Furthermore, chronic exposure to over-nutrition has been shown to promote abnormal immune responses and inflammation in a multitude of tissues, with pathological consequences—a phenomenon referred to as metabolically orchestrated inflammation or metaflammation (Hotamisligil, 2006). Although the primary focus of individual nutrients has been on fatty acids, cholesterol should also be considered in this context. Cholesterol has a fundamental role in almost every aspect of mammalian physiology, and misregulation of cholesterol metabolism results in toxicity both at the cellular and physiological level. Mammals are equipped with multiple regulatory mechanisms in

cells (i.e., the SREBP2 pathway) and in the circulation (i.e., LDL metabolism) that coordinate cholesterol synthesis, metabolism, storage, and distribution to constrain cholesterol levels within a very narrow range in cells, in distinct cellular compartments, and in the circulation (Goldstein et al., 2006; Tabas, 2002). However, this safety net can be disturbed by genetic abnormalities such as familial hypercholesterolemia or those that impair yet unknown cellular countermeasures, especially under poor dietary habits that promote excess cholesterol storage; this disruption can lead to the triggering of inappropriate immune responses and associated diseases such as atherosclerosis (Tabas, 2002; Tall and Yvan-Charvet, 2015). Ito et al. (2016) now provide insight into the cellular mechanisms underlying aberrant immune responses to misregulation of cholesterol handling. Their findings suggest that genetic or environmental factors that predispose people to excess cholesterol storage, specifically in lymphoid organs, may promote the pathogenesis of autoimmune diseases. The rationale underlying this investigation was based on an earlier study sug-

1186 Immunity 45, December 20, 2016 ª 2016 Elsevier Inc.

gesting that the transcriptional regulators liver X receptors (LXRs) lie at the interface between cholesterol metabolism and immune function, with a particular relevance to autoimmune disease (AGonzalez et al., 2009). In macrophages, LXRs sense oxysterols—a surrogate marker of cholesterol excess—and respond by promoting cholesterol efflux onto the lipid-poor apolipoprotein AI (ApoAI) particles, which comprises the high-density lipoproteins that mediate reverse transport of cholesterol out of the macrophage and into the liver for excretion. A-Gonzalez et al. (2009) found that LXR deficiency (genetic deletion of both LXRa and LXRb) results in diminished clearance of apoptotic cells by macrophages and autoimmunity associated with defects in T cell tolerance and the production of autoantibodies by B cells. The authors speculated that at least part of the anti-inflammatory function of LXR was due to its role in cholesterol efflux. However, the immune cells driving autoimmune disease upon LXR deficiency were not defined. It was also possible that LXR mediated this function by directly regulating immune-related gene targets, as opposed to the misregulation of cholesterol metabolism