A typical CD8 T cell response to primary infection with acute viral or intracellular bacterial pathogens is characterized by three distinct phases—expansion, contraction, and memory. Upon stimulation with cognate antigen in conjunction with costimulatory and inflammatory ligands, naïve cells undergo massive clonal expansion (up to 50,000-fold) and concomitant effector differentiation to generate large numbers of cytotoxic T lymphocytes (CTL), which serve to control the pathogen by migrating to peripheral sites of infection and elaborating cytotoxicity against infected target cells and producing effector cytokines such as IFN-γ and TNF-α (6–11). It is now well-established that the effector CTL pool broadly contains two distinct subsets—(1) short-lived effector cells (SLECs), which are fated to rapidly die after pathogen clearance, and (2) memory precursor effector cells (MPECs) (12–16), which are imprinted with antigen-independent survival capabilities for mediating long-term protection against secondary challenge (17–19). Thus, supporting the concept of memory programming, or imprinting of cardinal memory properties during primary expansion (20–23), several studies have now demonstrated that the balance of MPECs and SLECs can be altered by manipulating the duration of antigen, IL-2 and other inflammatory cytokine signals (14–16, 24). In fact, as discussed later, the heterogeneity of the memory CD8 T cell pool is likely programmed by differential signals accrued during the primary expansion phase. IL-2 signals (paracrine or autocrine) in particular exert crucial roles in effector and memory differentiation and function.

Optimal T cell activation with cognate peptide-MHC-I and costimulatory ligands result in IL-2 production and induction of IL-2Rα (CD25) expression, which along with IL-2Rβ (CD122, also used for IL-15 signaling), and IL-2Rγ (CD132, also referred to as common γ-chain as it is shared by other cytokines of the γ-chain family such as IL-4, 7, 9, 21) (5), forms the high affinity heterotrimeric receptor for robust IL-2 signal transduction and clonal expansion and effector differentiation (2). Much of the early work on IL-2 regulation of T cell responses relied on reductionist in vitro studies where amount and duration of TCR and IL-2 stimulation can be tightly controlled. These studies established a critical role for IL-2 as a T cell growth factor in driving cell cycle progression and expansion of CD8 T cells following TCR stimulation (25). Similar conclusions were reached following in vivo administration of IL-2, which engendered enhanced effector and memory pools of antigen-specific CD8 T cells (26–29). While these studies demonstrate that CD8 T cell differentiation events are amenable to manipulation by IL-2, physiological relevance of IL-2 in shaping a developing CD8 T cell response was uncovered following the development of Il2 germline-deleted mice. Studies in IL-2 knockout mice are confounded by Treg deficiency and associated spontaneous lymphoproliferative disease (30, 31). Hence, irreconcilably disparate outcomes of reduced or unaltered expansion and effector differentiation were reported in the context of infections and peptide immunization in IL-2 knockout mice (32–35). Nonetheless, bypassing pleiotropic immune effects in straight IL-2 and IL-2Rα (CD25) knockout mice, subsequent studies engaged the strategy of adoptively transferring IL-2- or IL-2Rα-deficient TCR transgenic CD8 T cells into wild-type recipients. In these studies, enumeration of antigen-specific CD8 T cells in an otherwise wild-type milieu using congenic differences without the need for restimulation, clearly established a requirement for IL-2 signals in driving optimal primary expansion of antigen-specific CD8 T cells in secondary lymphoid as well as non-lymphoid tissues (36, 37). IL-2 promotes effector differentiation through STAT-5-mediated Blimp-1-dependent induction of effector molecules (16, 38–42). In this regard, proinflammatory cytokine signals such as IL-12, IFN-γ, and type-1 interferons (IFN-α/β)—commonly referred to as signal 3 for their role in promoting optimal clonal expansion of effector CD8 T cells—are believed to complement IL-2, possibly non-redundantly (43, 44). Such collaboration, particularly between IL-12 and IL-2 has been recently shown to be important for optimal expression of transcription factors T-bet and Blimp-1, which synergize to drive a terminal effector differentiation program in CD8 T cells (45).

In addition to promoting CD8 T cell expansion and effector differentiation, IL-2 signals are also necessary for memory responses. IL-2Rα upregulation early after TCR stimulation is critical for formation of memory cells with robust secondary expansion capability (46, 47). Subsequent correlations of the duration of IL-2Rα expression with final memory outcome in a physiologically relevant setting—where the natural course of CD8 T cell response was not disturbed—revealed that rapid downregulation of IL-2Rα is equally important for memory development (16). Fate-tracking analyses showed that following an initial burst of IL-2 signals through IL-2Rα, curtailed expression of IL-2Rα and diminished IL-2 signaling is associated with memory fate, whereas prolonged expression of IL-2Rα and stronger IL-2 signaling drives terminal effector differentiation (16). Stronger IL-2 stimulation (100 U/ml) during in vitro priming also drives terminal differentiation compared to weaker signals (10 U/ml) (41). Similar findings have been reported in the DC-peptide immunization models as well as during murine infection with Lymphocytic choriomeningitis virus (LCMV), Listeria monocytogenes (LM), Vaccinia virus (VV), and Vesicular stomatitis virus (VSV) (16, 48). Moreover, constitutive activation of STAT-5 (key signal transducer of common γ-chain cytokines) also causes terminal differentiation (49). Consistent with the pro-proliferative role of IL-2, terminally differentiated effector CD8 T cells (SLECs) that express IL-2Rα for longer duration during an acute infection expand more than their memory-fated counterparts (MPECs) that downregulate the expression of IL-2Rα earlier (15, 16, 50–52). Together, these findings support the notion that metered IL-2 signals are required for optimal protective immunity and present a model of rheostatic control of CD8 T cell fates by IL-2 during acute infections.

All memory cells that survive after clearance of a primary infection are not created equal. Protective CD8 T cell immunity, as we understand it today, consists of collaborative defense against secondary challenge through concerted actions by a complex mixture of memory cells with distinct phenotypes, location, migratory properties, polyfunctionality, antigen-independent longevity, and potential for mounting rapid and robust clonal expansion and effector functions upon secondary challenge (44). As is expected from a spectrum of effector CTLs—that develop in response to varying doses and durations of antigen perceived in a variety of immune contexts, such as dose and duration of cytokines (e.g., IL-2, IFN-I, IL-12, IL-21, TGFβ, etc.), costimulatory signals, CD4 T cell interactions—a veritable spectrum of memory cells exist in a host after antigen clearance. At the risk of oversimplifying the CD8 T cell memory complexity, one can arguably categorize memory cells broadly into two major subsets—lymphoid or central memory (T_CM), and non-lymphoid memory, which is further distinguished into tissue-resident memory (T_RM), and migratory memory. Defined by their location, central memory cells largely recirculate through secondary lymphoid organs; tissue-resident memory (T_RM) cells—true to their name—set up permanent residence at front-lines of pathogen exposure; whereas migratory memory cells comprise a heterogeneous population that is capable of recirculation to peripheral tissues, and may be further distinguished by intravascular staining methodology into the CX3CR1^hi effector memory subset (T_EM) which does not enter extravascular space, and the less differentiated CX3CR1^int memory subset capable of migration into extravascular spaces (53, 54). T_RM cells serve effectively as the first line of defense against infections by virtue of their key properties of location at barrier sites and rapid elaboration of effector functions (cytotoxicity against infected target cells and effector cytokine production). Consistent with their ability to recirculate through peripheral tissues, T_EM cells retain higher expression of effector molecules, and are believed to aid T_RM cells in protecting against secondary challenge along with the extravascular migratory memory cells. In contrast, T_CM cells largely downregulate their effector program after antigen clearance, but are capable of rapid upregulation of the effector program upon antigenic rechallenge, also have superior polyfunctionality (ability to coproduce multiple cytokines such as IL-2, IFN-γ, and TNF-α), and expand more vigorously to aid the T_RM and migratory cells during secondary challenge.

Developmentally, fate-tracking experiments show that effector CD8 T cells that rapidly downregulate IL-2Rα largely give rise to central memory and effector memory cells. In comparison, effector CTLs, with prolonged IL-2Rα expression, largely give rise to terminal effector and effector memory fates; and curtailed stimulation of these cells by adoptive transfer into infection-controlled recipients (removal of antigen, IL-2, and all other infection-related signals) results in less terminal differentiation, as evidenced by increased proportions of effector memory cells compared to short-lived effector cells. These observations are consistent with a role for increasing IL-2 in driving effector CD8 T cells progressively toward terminal differentiation. It is believed that T_RM cells arise from relatively less differentiated memory precursors, which first seed the peripheral sites such as skin and small intestines (55, 56). In situ, the precursors receive microenvironment-specific developmental cues that drive the expression of unique chemokine receptors, integrins, and transcription factors for T_RM cell tissue residency and local protection (56–61). Within the tissue, the transforming growth factor β (TGF-β) exerts a critical role in directing the T_RM differentiation program in concert with other tissue-specific signals (55, 56, 62, 63). While CD8 T_RM cells capable of IL-2 production have been recently reported in skin and liver (64, 65), and IL-2 signals have been shown to be important for maintenance of allergic T_H2-type cells in the lungs (66), murine studies directed at understanding whether early IL-2 signals are necessary for T_RM seeding of tissue sites, whether prolonged IL-2 signals compromise T_RM cells, and how TGF-β signals and other tissue-specific factors work in conjunction with IL-2 signals (synergistically or antagonistically) to drive the differentiation, maintenance, and recall function of T_RM cells within the local sites remains to be fully explored. Likewise, whether similar rules of progressive terminal differentiation with increasing IL-2 signals are also active in situations of chronic antigen stimulation—as occurs during persistent viral infections and cancers—remains to be defined.

IL-2 couples T cell expansion and effector differentiation through induction of multiple downstream signaling cascades. Expression of pro-differentiation transcription factors, Blimp-1 (16, 38, 40–42) and Id-2 (83), is largely mediated through STAT-5 activation in response to IL-2 stimulation (2) (Figure 1). Reciprocal suppression by IL-2 of transcriptional factors that promote T cell memory such as Bcl-6 (41, 84–88) (which also represses Blimp-1 expression) is believed to further fix the terminal effector differentiation program (45). IL-2 is believed to regulate the expression of Bcl-6 through activation of Akt, which serves to control the activity of Foxo family transcription factors (89), Activation of Akt also alters the expression of proteins involved in CD8 T cell trafficking such as CD62L, CCR7, and S1P1, so as to promote their migration to peripheral sites of infection and inflammation (90–92). In addition to activation of STAT-5 and Akt, which largely promote effector differentiation, IL-2 links effector differentiation with clonal expansion through activation of MAPK signaling and T cell activation, cell cycle progression and survival programs (89). Sustained expression of cMYC through IL-2 drives proliferation by upregulating cyclins and anti-apoptotic molecule B-cell lymphoma 2 (Bcl-2), and by downregulating p21 (93, 94). In addition to cell cycle regulators, Myc also controls key metabolic aspects of T cell activation and proliferation (95). Myc promotes glycolysis and glutaminolysis through upregulation of key enzymatic and transporter proteins (96, 97). In this regard, mTOR also serves as a primary hub to integrate environmental cues from growth factors such as nutrients and IL-2 to promote glycolysis (94, 98, 99), oxidative phosphorylation and anabolic processes such as protein, lipid, and nucleotide biosynthesis necessary to sustain proliferation (96, 97). How effector and memory CD8 T cell fates are defined in vivo through differential metabolic programming by varying IL-2 strength or duration remains to be elucidated.

With diverse outcomes of memory and terminal effector differentiation in CD8 T cells that receive short/weak as opposed to prolonged/strong IL-2 signals (as described earlier), it remains to be determined whether rheostatic control of transcriptional and metabolic regulation occurs. It is plausible that curtailed or weak IL-2 signals drive lower levels of STAT-5, Akt and mTOR activity, thus resulting in lesser proliferation, effector differentiation and trafficking to peripheral sites of infection. In contrast, strong and prolonged IL-2 signals may drive stronger STAT-5, Akt, mTOR and MAPK activity, thus leading to augmented proliferation, effector differentiation and migration to peripheral sites of infection, where the microenvironmental niches further reinforce the terminal differentiation programs through induction of receptors for inflammatory cytokines such as IL-12 (100), and inhibition of IL-7 (101) receptor levels. Notably, a role for Tregs has been implicated in regulating the amount of IL-2 signals to memory-fated CD8 T cells by acting as IL-2 sinks (102) during CD8 T cell expansion. During later stages in the absence of antigen (when IL-2 is limiting) also, Tregs continue to curtail T cell stimulation and proliferation to maintain memory CD8 T cell quiescence through CTLA-4 (103) and IL-10 (104) inhibitory mechanisms and possibly through IL-2 restriction (105). IL-2 is also bound to the extracellular matrix through heparan sulfate moieties (106) to presumably increase local concentrations, thus supporting the notion that strong and prolonged IL-2 signals can be achieved in vivo. Effectually, quantal differences in IL-2 signals may lead to differences in signaling thresholds that ultimately result in terminal effector gene expression patterns driven by Blimp-1, T-bet, Id-2, and cMyc, or in memory lineage gene expression patterns characterized by augmented Bcl-6, Eomesodermin and Id-3. Indeed, analogous rheostatic control of CD4 T cell fates by differential levels of IL-2 signaling has been reported in the balance of T_H1 and T_FH fate determination (107, 108) through reciprocal regulation of T-bet and Bcl-6 by mTORC1-dependent control of the glycolysis gene expression program (109).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.