CTLs Get SMAD When Pathogens Tell Them Where to Go Free

Recent estimates from the World Health Organization indicate that infections with SARS-CoV-2 caused ∼15 million deaths within a period of ∼2 y (1). Many symptoms of severe disease are linked to a massive inflammatory response that develops in the lungs when innate immune cells undergo pyroptosis (2). Although hospitalization rates have been greatly reduced by vaccination, concerns about waning immunity underscore the importance of investing in strategies that elicit broad immunity to new variants (3). Although Ab-based immunity can be bolstered using vaccines that include T cell epitopes from conserved viral proteins (4–6), current methods of vaccination fail to capitalize on this type of protection largely due to difficulties in directing vaccine-induced memory CD8 T cells to the site of pathogen exposure. This review highlights several studies that expand our knowledge of the mechanisms that govern how CTLs respond to infection and mechanisms that promote localization in specific tissues.

CTLs express a variety of homing receptors that are dynamically regulated at specific stages of CD8 T cell differentiation during an infection. The fate and functional properties of the responding CTLs are determined by multiple factors, including the strength of signals from the Ag receptor (7), exposure to costimulatory molecules, cytokines, and metabolic mediators (8).

Under homeostatic conditions secondary lymphoid organs (SLOs) are surveyed by quiescent populations of naive CD8 T cells that circulate in the bloodstream. During infection, these cells differentiate into mixed populations of effector T cells (T_EFF) and memory CD8 T cells that perform different functions to generate an efficient immune response. Short-lived T_EFF are programmed for a rapid lytic response and have limited capacity for self-renewal and disappear as the infection resolves (9–12), whereas heterogeneous populations of memory CD8 T cells remain in the circulation and peripheral tissues. The surviving CTLs also include terminally differentiated memory cells that have been termed as long-lived effector cells, or terminal effector memory CD8 T (t-T_EM) cells that reside in the vasculature during the recovery phase of infection (13–15). For simplicity, we refer to these long-lived killer cell lectin-like receptor G1 (KLRG1)⁺ CTLs as terminal memory T cells (T_TM), and these cells will be discussed in more detail later in this review. Other blood-borne CTLs are effector memory T cells (T_EM) that maintain a partially activated phenotype and circulate through peripheral tissues, but are excluded from resting lymph nodes (16). Some T_EM enter reactive lymph nodes and destroy Ag-bearing dendritic cells (DCs) as homeostasis is restored (17). Within a few months the numbers of T_EM decline, as central memory T cells (T_CM) undergo homeostatic proliferation and become the dominant population of memory cells in the circulation (18). T_CM lack constitutive T_EFF functions and express adhesion molecules that are required to access peripheral lymph nodes under resting conditions. Peripheral lymph nodes also provide a survival niche for small numbers of long-lived stem-memory T cells (T_SM) that express stem cell Ag-1 (Sca1) (19–21). These multipotent T_SM are capable of robust proliferation and replenish the memory compartment upon secondary challenge.

Many different approaches have been used to examine the mechanisms that influence the functional properties of pathogen-specific CTLs. Some studies suggest that CTLs follow a linear differentiation pathway and exchange surface markers as they transition between subsets (22), whereas others indicate that phenotypically distinct subsets of CTLs develop simultaneously (23). Current data indicate that both mechanisms contribute to functional diversity as CTLs respond to infection. Fate-mapping techniques and limiting-dilution assays were used to show that a single naive CD8 T cell could produce a mixed population of T_EFF and memory CD8 T cells (24, 25). Others analyzed CTLs during cell proliferation and found that a single precursor could produce a T_EFF and a memory CD8 T cell simultaneously, as some genetic markers were not equally distributed between the daughter cells after the first round of cell division (26, 27). Transcriptome analyses also showed that naive T cells are equally poised toward T_EFF or memory formation after the first round of cell division (28). Recent evidence indicates that genetic programming of CTLs begins in the SLO, as some naive CD8 T cells are preconditioned to become resident memory T cells (T_RM) during their interactions with APCs that activate TGF-β (29).

Although some memory CD8 T cells circulate around the body, peripheral tissues are populated with noncirculating T_RM (29). T_RM reside in virtually all tissues of the body (30), including reactive lymph nodes (31), and they make important contributions to protective immunity (32). The demarcation between different subsets of memory cells blurs during chronic infection as exhausted CTLs are formed with upregulated inhibitory receptors (33, 34).

During recent years, technical advances have increased our knowledge of the mechanisms that govern how activated CTLs respond to infection. Many groups used intravascular staining to discriminate between CTLs that are located in the blood vessels and peripheral tissues, whereas others used parabiosis to identify CTLs that were sequestered from the circulation for an extended period of time (35, 36). The data show that heterogeneous populations of pathogen-specific CTLs can be broadly divided into two groups based on their tissue distribution (Fig. 1). The injected Abs preferentially label nomadic populations of CTLs that use the bloodstream to transit between different organs (Fig. 1A, 1C), whereas other CTLs are adapted for immune surveillance in peripheral tissues (Fig. 1B, 1C). To gain additional insights into the processes that influence the fate decisions of activated CTLs, we organized phenotypically distinct subsets of CTLs according to their homing characteristics. This pattern reveals a continuum of differentiation that begins when highly differentiated T_EFF enter the circulation during robust inflammation and are superseded by less differentiated subsets of memory CD8 T cells as homeostasis is restored.

Sphingosine 1-phosphate (S1P) is a lipid metabolite that plays a central role in leukocyte migration and is present at high concentrations in blood (37). CTLs express two S1PRs (S1PR1 and S1PR5) that belong to the G protein–coupled receptors family. S1PR-expressing CTLs follow S1P gradients in the blood while migrating to different tissues. S1PR1 is required for circulating CTLs to transit through SLOs (38), whereas S1PR5 is required for CTLs to emigrate from peripheral tissues (39). Temporal changes in S1PR expression determine whether CTLs enter the bloodstream or remain sequestered in the local tissues. S1PR1 and S1PR5 are regulated via different mechanisms. S1PR5 is downregulated by TGF-β to prevent T_RM from entering the circulation (39), whereas S1PR1 is downregulated through its interactions with CD69 (40). CD69 is a transmembrane protein that is transiently expressed on newly activated CTLs after Ag stimulation. As these CTLs undergo clonal expansion, the transmembrane region of CD69 interacts with S1PR1 to form complexes that are internalized to impede cell migration (40, 41). CD69 expression gets downregulated as T_CM circulate around the body, whereas CD69⁺ T_EFF and T_RM extravasate to peripheral tissues (31, 42).

Circulating leukocytes are recruited from the blood vessels by inflammatory molecules that are released in infected tissues. Proinflammatory cytokines increase vascular permeability and initiate an adhesion cascade that is required for transendothelial migration (43, 44). The extravasating cells include a mixed population of T_EFF and T_RM that express CD69. Most T_EFF disappear as the infection comes under control, whereas other cells become noncirculating T_RM that reside in the local tissues (45). For several months, T_RM are the largest population of pathogen-specific CTLs in the body (46), and the numbers increase during exposure to diverse environmental pathogens (47). These cells remain near the sites of pathogen exposure where they can provide rapid responses upon reinfection (32). Because the requirements for tissue retention and cell migration vary between different tissues, this review primarily focuses on mechanisms that are controlled by TGF-β.

TGF-β is a pleiotropic cytokine with diverse regulatory functions. A large reservoir of latent TGF-β is maintained in peripheral tissues, where the latency-associated peptide present in TGF-β attaches it to the extracellular matrix (48, 49). Platelets express a docking receptor called glycoprotein A repetitions predominant (GARP) that retains latent TGF-β in the blood supply (50). During infection, active TGF-β is released from the reservoir by integrins and proteases that cleave latency-associated peptides (48, 49). The active domain of TGF-β is a charged peptide with a short half-life (51, 52) that limits the sphere of activity to specific anatomical niches (53).

TGF-β plays an important role in regulating the expression of several homing receptors in activated CTLs such as L-selectin (CD62L) (54, 55) and cadherin-binding proteins such as CD103 (α_Eβ₇ integrin), which interacts with epithelial cadherin (E-cadherin) (56) and KLRG1, which binds to several members of the cadherin family (57, 58). CD103 is expressed on naive CD8 T cells and large numbers of T_RM in peripheral tissues during chronic stimulation with TGF-β (31, 42). This molecule augments immunity by facilitating retention of T_RM at barrier surfaces and in the tumor microenvironment where cells express E-cadherin (59, 60). In addition to TGF-β, tissue retention is also facilitated by ICOS signaling in the local tissues (61). TGF-β regulates the activities of T_RM by increasing dependency on common γ-chain cytokines (IL-7 and IL-15), thereby suppressing homeostatic cell division (62). At the same time, TGF-β enhances T_RM survival in tissues that have low oxygen concentrations by upregulating hypoxia-inducible factors (63). These data indicate that TGF-β plays an important role in differentiation, proliferation, and maintenance of T_RM in nonlymphoid peripheral tissues.

TGF-β family members elicit cellular responses via a network of canonical and noncanonical signaling pathways (64). Canonical signals are mediated by group of structurally related molecules known as SMAD proteins (65). During signaling, receptor-regulated SMAD proteins (R-SMADs) are phosphorylated by multisubunit receptors and create multimeric complexes that enter the nucleus for gene regulation (66). TGF-β and activins signal via R-SMAD2/3, whereas bone morphogenic proteins and related growth differentiation factors signal via R-SMAD1/5/8 (66). SMAD4 facilitates signaling by both groups of cytokines and chaperone complexes of phosphorylated R-SMADs into the nucleus (66).

Several groups used Cre/lox recombination to examine the regulatory functions of TGF-β during T cell development. When CD4-Cre was used to prevent TGF-β receptor II (TGF-βRII) expression, it induced spontaneous T cell activation leading to severe autoimmune disease in mice, whereas no immune pathology was detected when TGF-βRII was ablated using the distal Lck promoter (dLck) (67). Because CD4 expression happens at early stages of T cell development, CD4-Cre–mediated gene ablation of TGF-βRII caused abnormal development of T cells, revealing a role for TGF-β signaling in this process. On the contrary, dLck-mediated Cre recombination happens at later stages of development after TCR rearrangement, and when we used dLck-Cre to prevent SMAD4 expression in antiviral CTLs during influenza A virus (IAV) infection, we observed normal T cell priming and proliferation. SMAD4-deficient CTLs also produced IFN-γ and TNF-α in levels similar to those in wild-types. We found that very few SMAD4-deficient CTLs expressed KLRG1 or CD62L and CD103 was expressed in the both the spleen and lungs (68). This indicates that SMAD4 is required for the formation of T_EFF and T_CM, and not for T_RM (68). Others observed similar phenotypic changes when they used Cre recombinase driven by the proximal Lck promoter to prevent SMAD4 expression during infection with Listeria monocytogenes (69). The timing of gene ablation was important, as SMAD4-deficient CTLs showed altered CD44 expression and impaired cytolytic activity while using proximal Lck-Cre (69). The phenotypic changes that occurred after SMAD4 ablation were unexpected, as antiviral CTLs that lacked the TGF-β receptor displayed a reciprocal phenotype (68, 69). To study these signaling pathways in more detail, we generated CTLs with dual mutations. Infections with two different pathogens showed that SMAD4-deficient CTLs displayed consistently similar phenotypes in the presence and absence of TGF-βRII (55, 70). When we analyzed pathogen-specific CTLs for transcriptional changes, we found that SMAD4 and TGF-β regulated the same genes in opposite directions (55). In mice with dual mutations driven by CD4-Cre, SMAD4 ablation prevented autoimmune disease caused by CD4-Cre–mediated deletion of TGF-βRII (71). In each case, signaling via SMAD4 altered the properties of activated CTLs via a TGF-β–independent mechanism (55, 70, 71). Conventionally, TGF-β triggers the downstream signaling pathway through formation of the R-SMADs and SMAD4 trimeric complex. The signaling has even been shown to remain intact in some cancer cell lines that are deficient in SMAD4 (72). Surprisingly, in CD8 T cells, SMAD4 is functioning independent of TGF-β and importantly it is also actively counteracting the effect of TGF-β.

KLRG1 is an inhibitory receptor, with an ITIM in the cytoplasmic tail (58, 73, 74). table KLRG1 expression identifies terminally differentiated T_EFF that are programmed for robust lytic activity and short-term survival. T_TM that maintain KLRG1 expression have been detected during the recovery phase of infection with multiple pathogens, including IAV, lymphocytic choriomeningitis virus (LCMV), and L. monocytogenes (15, 68, 75, 76). These CTLs are of considerable interest, as intravascular staining indicates that they are adapted for localization in the vasculature (55, 68).

Postinfection with L. monocytogenes, the red pulp of the spleen contained T_TM that survived for several months and provided robust protection against reinfection (15). When analyzed for effector functions, these cells were primed for cytolysis, cytokine production, and weak proliferation (13). These CTLs develop from KLRG1⁺ precursors (13), whereas the precursors of T_CM and T_EM lack KLRG1 expression (13, 77). The size of the T_TM population increased during boosting, indicating that additional CTLs upregulated KLRG1 upon repeated Ag challenge. These long-lived KLRG1⁺ T_TM were also found in the spleen postinfection with LCMV (14). Single-cell RNA sequencing showed that the transcription profile of T_TM was distinct from other memory subsets (14). Selected subsets of genes that are associated with T_EFF functions remained elevated at 55 d postinfection (perforin and granzyme B), whereas proinflammatory cytokines (IFN-γ and TNF-α) were downregulated (27, 28). CTLs with similar characteristics were present in human blood from healthy donors (14). KLRG1⁺ CTLs have also been detected in highly vascularized nonlymphoid tissues such as the lungs (76). These CTLs persisted for more than a month after IAV infection and responded to secondary challenge after transfer (76). When analyzed by intravascular staining at 40 d postinfection, 99% of KLRG1⁺ CTLs were located inside the blood vessels (68).

KLRG1 interacts with two members of the cadherin family. Neural cadherin (N-cadherin) is expressed on endothelial cells and pericytes as well as some stromal progenitor cells (78, 79), whereas E-cadherin is expressed on inflammatory DCs and epithelial cells (80, 81). The epithelial-to-mesenchymal transition is a process that is induced by TGF-β during tissue repair (82). A cadherin switch occurs during the epithelial-to-mesenchymal transition as TGF-β induces N-cadherin while downregulating E-cadherin expression (83, 84). The function of KLRG1 is poorly understood and may involve cell migration or tissue retention. However, a recent study found that KLRG1⁺ CTLs colocalized with N-cadherin–expressing cells in the spleen after LCMV infection, indicating a role in the maintenance of memory CD8 T cells (85). Further work is required to determine whether KLRG1⁺ CTLs extravasate to infected tissues upon reactivation or dispense effector molecules inside the blood vessels. CTLs that reside inside the blood vessels may be strategically positioned to facilitate recruitment of circulating immune cells to infected tissues.

Fractalkine (CX₃CL1) is a chemoattractant that promotes T cell migration (86). Several studies used reporter mice to monitor changes in fractalkine receptor (CX₃CR1) expression (87) during infection and identified three subsets of pathogen-specific CTLs that expressed CX₃CR1 at different levels (88–90). The data showed that terminally differentiated T_EFF expressed KLRG1 and CX₃CR1 at high levels, whereas both markers are absent from T_CM and T_EM (86, 90). Lymphoid and peripheral tissues contained a third subset of CD62L⁺ memory CD8 T cells that expressed CX₃CR1 at intermediate levels (CX₃CR1^int). Although CX₃CR1^int CTLs were capable of robust proliferation and self-renewal, CX₃CR1 was downregulated by 8 mo postinfection, indicating that the memory population changed with time (89). Similar cells were detected when mice were infected with murine CMV, as CTLs in the inflationary pool displayed a CX₃CR1^int phenotype (88). However, it is not clear how the expression of CX₃CR1 is regulated in different subsets of CTLs. Using CX₃CR1-GFP reporter mice, we identified that very few SMAD4-deficient CTLs expressed KLRG1 or CX₃CR1 at high levels during L. monocytogenes infection (55). Several studies show that these CTLs localize in the vasculature and are thus implicated for a role in vascular surveillance (55, 68). Although signaling via SMAD4 plays a role in the formation of T_TM, further work is required to determine how these cells augment immunity and whether KLRG1 and CX₃CR1 influence their tissue distribution.

Although TcR stimulation induces multiple rounds of cell division, additional stimuli are required to produce fully differentiated CTLs. The inflammatory milieu that develops during infection varies according to the type of pathogen. When clonal populations of Ag-specific CTLs were used to analyze homing receptor expression during infections with different pathogens, the population dynamics reflected the local inflammatory environment (91).

Type I IFN (IFN-α and IFN-β) and IL-12 are proinflammatory cytokines with important functions during CD8 T cell differentiation (9, 11, 92). In prior studies, mice that lack the receptors for IL-12 and IFN-I were used to assess the requirements for cytokine stimulation during formation of terminally differentiated T_EFF. Variable results were obtained postinfection with different pathogens (93). Both cytokines were required for formation of KLRG1⁺ CTLs during infection with vesicular stomatitis virus, whereas IL-12 was sufficient to induce KLRG1 expression during infection with L. monocytogenes (94). However, during infection with vaccinia virus, these cytokines had little influence on T_EFF formation, but they were required for memory formation (93). Interestingly, rIL-12 alone did not induce KLRG1 expression on CTLs in vitro (95), indicating that additional stimuli are required for terminal differentiation.

Many cytokines that influence fate decisions of pathogen-specific CTLs signal via JAK-STAT pathways. During T_EFF formation, IL-12 signals via STAT4 (96) and IL-2 signals via STAT5 (97). Postinfection with vaccinia virus, IFN-I enhanced CD8 T cell survival via a STAT1-dependent mechanism (98), whereas STAT3-dependent signaling by IL-10 and IL-21 was required for the maintenance of memory CD8 T cells postinfection with LCMV (99). IL-2 and IL-21 are members of the common γ-chain family of cytokines that also support T_EFF formation (100). KLRG1⁺ CTLs acquire cytolytic activity after IL-12 upregulates the IL-2 receptor (CD25) and T-bet (11), whereas IL-21 upregulates the transcription factor basic leucine zipper ATF-like transcription factor (BATF), a transcription factor that favors T_EFF formation (101). When IL-21R–deficient mice were infected with microsporidia Encephalitozoon cuniculi, only small numbers of T_EFF expressed KLRG1 (102). These data indicate that in addition to IL-12 and type I IFN, IL-21 also functions toward inducing the differentiation of KLRG1⁺ CTLs.

Four pairs of transcription factors act at the bifurcation between T_EFF formation and memory precursors, including T-bet/Eomesodermin (EOMES) (103, 104), zinc finger E-box binding homeobox (ZEB)1/ZEB2 (105), Blimp1/Bcl-6 (106), and Id2/Id3 (107, 108). Cytokines adjust the ratios of these transcription factors as CTLs respond to infection. ZEB proteins play important roles in hematopoiesis and cellular plasticity (109). ZEB2 is expressed at an early stage during CD8 T cell differentiation and cooperates with T-bet to induce formation of terminally differentiated T_EFF (110). During memory formation, TGF-β and miR-200 downregulate ZEB2 and induce ZEB1 expression (105). At the same time, T-bet is downregulated by runt-related transcription factor 3 (Runx3) (111).

A network of transcription factors is required to generate terminally differentiated T_EFF, including IFN regulatory factor 4 (IRF4) (112, 113) and BATF, which cooperatively regulate T-bet, Runx3, and Blimp-1 expression (114). During T_EFF formation, BATF and IRF4 upregulate the IL-12 receptor and Blimp1, while silencing genes that support memory formation such as the IL-2 receptor (CD25) and CD27 (115). Additionally, there are other transcription factors that inhibit T_EFF formation to program CTLs toward memory or exhaustion. Notably, Bach2 restrains T_EFF formation by limiting accessibility of AP-1 binding sites (116). Nr4a1 (Nur77) and Fli1 inhibit the expression of IRF4 (117) and Runx3 (118), respectively, to limit T_EFF differentiation. During chronic infections, Blimp-1 promoted the expression of inhibitory receptors (PD1 and LAG3), the hallmark features of T cell exhaustion (119). T cell factor 1 (TCF1) is part of the canonical Wnt signaling pathway and plays an important role during CD8 T cell differentiation. TCF1 promotes the formation of T_CM and is also expressed at high levels in T_SM. In addition, it augments T_EFF functions during chronic viral infections by upregulating several transcription factors, including EOMES (120, 121). However, in activated CTLs it binds to the itgae gene, inhibiting TGF-β–mediated CD103 expression, and there is a rapid decrease in TCF1 expression during T_RM development (122).

IL-12 and TGF-β cooperatively regulate genes that support memory formation, including the T-box transcription factors T-bet and EOMES (62, 104, 123, 124). During infection, IL-12 supports T_EFF formation by repressing EOMES and inducing T-bet expression (123). Similarly, EOMES is downregulated by TGF-β during T_RM development in peripheral tissues but is expressed in increased levels in T_CM (54, 124). Homolog of Blimp-1 in T cells (HOBIT) is a transcription factor that is involved in formation of T_RM (125). EOMES has an important role in delineation of circulating and noncirculating CTLs, as a recent study found that EOMES binds to the Hobit loci to suppress T_RM differentiation (126). Our data show that SMAD4 binds to the EOMES promoter and positively regulates its gene expression, which in turn inhibits T_RM development and downregulates CD103 (55, 124). SMAD4 and TGF-β are essential components of two independent signaling pathways (55, 70, 71) that cooperatively regulate homing receptors by adjusting the expression levels of the same genes in reciprocal directions (55). Genes that are regulated by SMAD4 and TGF-β include homing receptors such as Klrg1, Sell (CD62L), and Itgae (CD103) and transcription factors such as Hobit and Eomes (55).

Epigenetic modifications in key genes, through DNA methylation and/or histone modification, dictate the fates of activated CTLs. Histones are positively charged proteins that organize genomic DNA into densely packed structures known as nucleosomes. Chromatin remodeling controls gene expression by exposing (or masking) regulatory sequences in the genomic DNA. As pathogen-specific CTLs undergo clonal expansion, the chromatin landscape is altered by lineage-specific transcription factors for expression of genes that control T_EFF functions, proliferation, and survival (127, 128).

Genes can be silenced by methylation of CpG islands in the promoter region, or modification of histones by methylation or acetylation. In naive CD8 T cells, key genes that are poised for activation carry bivalent chromatin modifications that include permissive (H3K4me3) and suppressive (H3K27me3) (129, 130) histone marks. During Ag stimulation, repressive marks are removed from genes that encode T_EFF functions, such as ifng and tbx21 (T-bet), whereas permissive marks (H3K9Ac and H3K4me3) are deposited to activate genes that support effector function (131). Proinflammatory cytokines (IL-12 and IFN-I) alter the differentiation program of pathogen-specific CTLs by hyper-acetylating histones in the loci that encode Eomes and Granzyme-B (12).

Polycomb repressive complex 2 (PRC2) is a protein complex that modifies lysine residues on histones and alters the epigenetic landscape of T cells during activation and proliferation (132). Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the PRC2 complex with methyltransferase activity. During infection with LCMV, inactivation of the PRC2 complex prevented H3K27me3 modifications in memory-associated genes (Tcf7, KLF2, and SMAD2) (28, 132). Notably, EZH2-mediated histone modifications were blocked during memory formation by the transcription factor forkhead box O1 (FOXO-1) (132). During T_EFF formation, the genes that encode the IL-7 receptor and CD62L are silenced by the histone methyl transferase Suv39h1. During infection with L. monocytogenes, deletion of Suv39h1 prolonged memory survival and impaired pathogen clearance (133). Bromodomain protein 4 (BRD4) is a chromatin modifier that binds to acetylated lysine residues in enhancer regions and sustains transcriptional activity during terminal differentiation (134). Recent studies identified that protein arginine methyl-transferase 5 (PRMT5) blocks T_EFF formation by modifying the loci for Blimp1 using novel repressive marks where the arginine residue gets methylated instead of lysine (H4R3me2 and H4R8me2) (135).

DNA methyl transferase 3a (Dnmt3a) activity increases during T cell activation and supports memory formation by silencing T_EFF genes (136). As naive CD8 T cells become T_EFF, genes that encode T_EFF functions (ifng, granzyme B, granzyme K, and perforin) are rapidly demethylated (135, 137, 138). Ten-eleven translocation protein 2 (TET2) is a dioxygenase that is induced by IL-12 and demethylates the ifng loci expression during LCMV infection (139). Memory-associated genes such as Sell, Tcf 7, and Ccr7 are regulated by similar mechanisms during Ag stimulation (140). During LCMV infection, loss of Dnmt3a activity resulted in accelerated development of T_CM that expressed CD62L and CD127 (140). Similarities between the epigenetic signatures of T_CM, T_EM, and T_SM suggest that key genes acquire epigenetic marks progressively modified during differentiation (141). Supporting the previous study, whole-genome bisulfite sequencing of human naive and memory cells showed that in memory subsets the effector gene loci were demethylated and methylation status was unaffected during IL-7– and IL-15–mediated proliferation (142).

TGF-β and SMAD4 have important functions during formation of pathogen-specific CTLs that localize in different regions of the body. In thymus, conditioning by TGF-β is required to produce quiescent populations of naive CD8 T cells that circulate through SLOs. After Ag stimulation, naive CD8 T cells differentiate into multiple subsets of pathogen-specific CTLs that perform different functions during the immune response. The migration patterns of CTLs are influenced by costimulatory signals and proinflammatory cytokines that are present in the local environment. APCs play a major role in T_RM development by activating TGF-β and provide costimulatory signals via ICOS. Recent studies show that SMAD4 is part of an alternative signaling mechanism that promotes formation of T_EFF and T_CM, as well as T_TM, that reside in the vasculature. Genes that are induced by SMAD4 include homing receptors that support a nomadic lifestyle (KLRG1, CX₃CR1, and Sell) and EOMES. Further work will reveal whether signaling via SMAD4 serves as a catalyst of lineage commitment by inducing epigenetic modifications during terminal differentiation.

The authors have no financial conflicts of interest.