The discovery of memory Treg raises some obvious questions: what are the evolutionary target-antigens of mTreg cells? In other words, which antigens are expressed/present intermittently and thus require regulatory memory to last between exposures? Although most self-antigens are likely persistently expressed,some, such as proteins in female breast milk, pregnancy-related antigens, and fetal antigens are encountered intermittently. In these cases, the initial expression of the neo-self-antigen or fetal antigens could recruit antigen-specific Treg cells to the respective tissue (i.e., breast, uterus) to then dampen any inflammation upon reencounter of the antigen in the tissue. This mechanism would increase the success of subsequent pregnancies. Thus, one could speculate that mTreg-specific for intermittently expressed antigens are a mechanism devised to face the challenges and changes that accompany sexual reproduction in mammals. In this way, mTreg are similar to pTreg, which seem to have evolved to mitigate the maternal–fetal conflict (152). Subsequently, both regulatory cell types may evolutionarily have been adapted to mediate microbiota-specific tolerance (33). Microbial antigens present at body surfaces may also be antigens we are only exposed to intermittently depending on changes in the respective flora and on breaches of the epithelial surface of skin or gut that result in increased release of microbial antigens. Thus, mTreg cells may be a useful mechanism to avoid inflammation in response to spikes in exposure to normal microbial flora at body surfaces. Other examples of intermittent antigen-exposure that may require regulatory memory are food antigens and allergens that the gut and skin are exposed to. The existence of allergen-specific mTreg in healthy individuals has not been formally shown but the success of allergen-specific immunotherapy relies on the induction of specific Treg cells that persist over long periods of time (153).

Which tissues/situations are amenable to the induction and maintenance of mTreg? Organs with environmental surfaces such as the skin, gut, and lungs have the highest likelihood of a barrier breach, and therefore, one might hypothesize that these organs have a battalion of self-reactive (and/or microbiota-specific?) mTreg cells positioned to prevent excessive inflammation and tissue damage in case of barrier breach. Indeed, this may have driven the ability of certain epithelial tissues to support mTreg cell maintenance (discussed further below). Additionally, it is possible that the regenerative capacity of a tissue is crucial for the development of mTreg cells. mTreg cells only make sense in tissues that can recover after inflammatory damage. Relatively, non-regenerative tissues such as the pancreas are perhaps less likely to harbor mTreg cells since the pancreatic islets are destroyed in the inflammatory response in type-1 diabetes and regulatory memory would not serve subsequent organ protection. In this context, it is possible that tissue stem cells instruct regulatory memory formation to allow faster regeneration in future inflammatory settings. Indeed, the immunomodulatory potential of stem cells, and in particular mesenchymal stem cells (MSCs), has been studied extensively in recent years. MSCs are pluripotent cells that are present in multiple tissues, including bone marrow, adipose tissue, skin, muscle, blood, and placenta (154). MSCs were shown to induce Treg cells *in vitro* via their production of prostaglandin E(2) and TGF-β (155). Additionally, they modulate their environment by secretion of mediators such as IDO and IL-10. Due to their immunomodulatory functions, numerous clinical studies using MSCs are currently underway to treat inflammatory diseases such as graft-versushost disease and autoimmunity (156). Stem cells have potentially evolved their ability to induce Treg cells because they seem to require them for their maintenance. For instance, Treg cells are attracted to the bone marrow by the stem cell chemoattractant

CXCL12 (SDF-1) (157). This localization to the niche was crucial for the preservation of the hematopoietic stem-cell niche in the bone marrow as Treg cell depletion resulted in a loss of allohematopoietic stem cells (158). Thus, Treg cells (and specifically mTreg cells) are potentially involved in preserving stem-cell niches from immune attacks and this may be one way in which they provide critical tissue-support functions.

#### **CONTROL OF Treg CELL MAINTENANCE**

Due to their potent immunosuppressive function, manipulation of Treg cell abundance is an attractive therapeutic strategy to either boost or inhibit immune responses in a variety of clinical settings (159). However, competition for growth and survival factors acts to limit the size of the Treg cell pool *in vivo*, and as a result clinical trials of adoptive Treg cell therapy have failed to achieve long-term cell engraftment or substantial clinical benefit (79). Although work over the last 10 years has defined several factors that help regulate Treg cell homeostasis, an integrated model of how Treg cell abundance, function, and distribution is controlled during normal and pathological immune responses is still lacking. A better understanding of the mechanisms regulating the abundance of different Treg cell populations is crucial for developing therapies to boost their activity to treat autoimmunity and prevent graft rejection, or to inhibit Treg cells in the contexts of cancer and chronic infection.

In conventional CD8+ and CD4+Foxp3+ effector T cells, it has become clear that different populations of naïve, effector, and memory T cells have distinct homeostatic requirements, and that this helps preserve the functional diversity of effector and memory T cells while ensuring that an adequate pool of naïve T cells is maintained in order to respond to new threats (160). This is in large part due to changes in the requirements that these cells have for different cytokines that signal through receptors utilizing the γc receptor subunit such as IL-2, IL-7, and IL-15. That Treg cells occupied their own homeostatic niches was apparent from early experiments in which Treg cells underwent robust population expansion and ameliorated autoimmune disease development when transferred into Foxp3 mutant mice lacking endogenous Treg cells (9). A similar niche-filling capacity of Treg cells is observed when Treg cells are acutely depleted (161). That Treg cells could be sub-divided into populations with different homeostatic behaviors (and therefore likely subject to distinct sets of proliferative and survival signals) has been appreciated for some time (82). However, the precise nature of these homeostatic niches remains poorly understood.

Consistent with their constitutive expression of the highaffinity IL-2 receptor component CD25, it has become clear that IL-2 plays a central role in Treg cell function and homeostasis. Accordingly, defects in IL-2 or various components of the IL-2 receptor lead to development of autoimmune/inflammatory diseases associated with Treg cell dysfunction. Treg cells themselves do not produce IL-2, and instead are stimulated in a paracrine fashion by IL-2 produced by activated conventional T cells (162). Through regulation of anti-apoptotic proteins such as Bcl-2 and Mcl-1, IL-2 can deliver potent survival signals to Treg cells (161, 163). Additionally, IL-2 can potently drive Treg cell proliferation, especially when present in excess during niche-filling or when administered as super-agonistic IL-2/α-IL-2 immune complexes

(161, 164). Paradoxically, after the identification of Foxp3 as a molecular marker of Treg cells it became apparent that the numerical deficiency in peripheral Treg cells in peripheral tissues in the absence of IL-2 signaling is relatively mild (165, 166), and correcting these deficiencies by knocking out the pro-apoptotic factor Bim failed to restore full Treg cell function in IL-2-deficient mice (167). Taken together, these data indicate that maintenance of at least some Treg cell populations is IL-2-independent, and that the effects of IL-2 on Treg cell function *in vivo* are more qualitative rather than quantitative. Indeed, a recent analysis of IL-2 signaling in Treg cells demonstrated that rather than acting as a trophic factor for all Treg cells, IL-2 signaling *in vivo* is largely restricted to "central" Treg cells that access sites of paracrine IL-2 production in the secondary lymphoid tissues via expression of the chemokine receptor CCR7 (25). This quiescent population of Treg cells was particularly sensitive to genetic or antibody-mediated blockade of IL-2 signaling, whereas rapidly proliferating "effector" Treg cells were effectively maintained in the absence of IL-2. IL-2 also promotes specific effector functions in Treg cells such as expression of CTLA-4 (167). As CTLA-4 expression by Treg cells in secondary lymphoid organs help prevent the initial activation and differentiation of autoreactive cells, selective loss of these central Treg cells helps explain why autoimmunity develops in the absence of IL-2 or CD25 despite the presence of effector Treg cells with at least some functional capacity.

In contrast to the IL-2-dependent central Treg cells, the abundance of CCR7− effector Treg cells is most profoundly influenced by signals through the TCR and associated co-stimulatory receptors such as CD28 and ICOS. That effector Treg cells compete for access to these signals is indicated by the fact that abundance of these cells is intimately linked to the number of antigen-presenting DCs (125). Moreover, the fact that DC-mediated Treg cell population expansion occurred even when IL-2 signaling was blocked indicates that signals through either the TCR or IL-2 act in separate pathways to control Treg cell abundance (25, 168). Consistent with this, although IL-2 signaling was not associated with Treg cell proliferation in central Treg cells, the high rate of homeostatic proliferation of effector Treg cells was completely dependent on continued TCR signaling (168). Moreover, effector Treg cells have a CD25loBcl-2loMcl-1lo phenotype indicative of IL-2 deprivation, and accordingly are highly apoptotic. Thus, after losing access to IL-2-dependent survival signals in secondary lymphoid tissues, effector Treg cells in non-lymphoid organs appear to balance rapid TCR-dependent cell proliferation with a high-rate of apoptotic cell death to maintain their steady-state abundance. In tissues such as the intestines, this creates a largely self-renewing Treg cell pool specific for local antigens that are effectively maintained despite low levels of cellular immigration (25).

Among the co-stimulatory receptors, loss of CD28 has the most dramatic impact of Treg cell abundance (169). However, this may be largely due to defective Treg cell development in the thymus as deleting CD28 specifically in Treg cells after their development did not recapitulate this phenotype, although the CD28-deficient Treg cells were functionally impaired (170). However, blockade of ICOS signaling causes a rapid decline in the abundance of effector Treg cells *in vivo* (25), and this can accelerate development of organ-specific autoimmune disease (171). Interestingly, this was not associated with defects in effector Treg cell proliferation, indicating that ICOS signaling may regulate effector Treg cell survival, perhaps through engagement of the PI3K/Akt signaling pathway.

The dependence of effector Treg cells on TCR and costimulatory signals, and their competition for access to DCs raises the possibility that effector Treg cells exist in multiple TCRdependent "micro-niches" as was recently described for conventional CD4+ T cells (92). In this scenario, Treg cells specific for any given autoantigen must compete with one another for access to antigen-bearing DCs, thereby linking the abundance of any given Treg cell specificity to the amount of autoantigen presented, and ensuring that a diverse TCR repertoire is maintained in effector Treg cells. Indeed, this is consistent with the data demonstrating that particular TCR specificities are enriched in specific tissue sites (33, 53).

Unlike effector Treg cells that appear to depend on continued TCR and co-stimulatory signals for their maintenance, memory Treg cells can reside in non-lymphoid tissues such as the skin for extended periods in the absence of continued antigenreceptor signaling, raising the question of how these populations are maintained (44, 59). Additionally, memory Treg cells displayed a high-rate of homeostatic proliferation even after antigen withdrawal (59). The continued proliferation and thus maintenance of memory Treg cells may be a consequence of their not requiring many of the signals thought to be essential for the responses of effector T cells, such as Akt and mTOR and becoming relatively independent of TCR-signals after initial activation (172). Additionally, in the absence of the continued TCR and co-stimulatory receptor signals that maintain effector Treg cells, it is likely that memory Treg cells rely instead on specific cytokine signals for their homeostatic maintenance. Surprisingly, although IL-2 was required for the development of memory Treg cells from naïve precursors in the secondary lymphoid tissues, memory Treg cells in the skin showed decreased CD25 expression and maintenance of these cutaneous cells was IL-2-independent. However, IL-7 receptor expression was dramatically upregulated on these cells, and blockade of IL-7R signaling resulted in the loss of memory Treg cells in the skin but not the skin-draining lymph node (49).

That maintenance of mTreg cells in the skin is IL-7-dependent raises the important question of how expression of IL-7R is regulated in these cells in such a tissue-specific manner. Expression of IL-7R in conventional T cells is controlled in large part by the transcription factor, Foxo1, which in T cells is inactivated and removed from the nucleus after phosphorylation by activated Akt following TCR stimulation (173). However, despite the fact that Treg cells rely on continued Foxo1 activity for their suppressive function, most Treg cells in secondary lymphoid organs express low levels of IL-7R (49, 174). This prevents Treg cells from competing with conventional naïve and memory T cells for access to IL-7 produced by stromal cells in these tissues, and implies that IL-7R expression is differentially regulated in conventional T cells and Treg cells. By contrast, antigen-specific Treg cells in murine skin uniformly expressed high levels of IL-7R both in the presence or absence of antigen expression (Iris K. Gratz, unpublished observations). Although the tissue-specific signals directing IL-7R expression by Treg cells in the skin have not been identified, this results in the maintenance of a stable population of tissue-resident mTreg cells

even in the absence of continued antigen or IL-2. However, the importance of IL-7 in the maintenance of memory Treg cells in tissues other than the skin has not been examined. For instance, due to their expression of CD122 and the γc chain, Treg cells are equipped to respond to IL-15 trans-presented on the surface of IL-15/IL-15Rα expressing cells, and rather than IL-7, this may help maintain mTreg cells in tissues rich in IL-15 such as the intestine.

Collectively, these recent data support the concept that rather than occupying a single homeostatic niche, multiple pathways of homeostatic maintenance exist for distinct populations of Treg cells in different tissue sites. These include IL-2-dependent maintenance of central Treg cells in secondary lymphoid organs, TCR/ICOS-dependent maintenance of effector Treg cells in inflamed non-lymphoid tissues, and IL-7-dependent maintenance of memory Treg cells in the skin (**Figure 2**).

# **CLINICAL IMPLICATIONS**

Therapies to prevent allograft rejection or treat autoimmune diseases have long relied on general immunosuppression using broadly acting and non-specific medications. Treg cells represent a promising new avenue with the possibility of long-lived and antigen-specific tolerance to self- or foreign-antigens. Reported clinical trials have focused on the expansion of Treg cells*in vivo* and *ex vivo* (175). Applications of both polyclonally expanded Tregpopulations and antigen-specific Treg cells are currently moving into the clinic, and results have thus far shown acceptable safety and promising efficacy of the treatment (176). Conversely, inhibition of Treg cell function may enhance immunotherapy to cancer, and help promote resolution of chronic infection.

Resolution of the particular issues of Treg cell biology raised in the above will certainly help in the targeted development of Treg cell-based therapies. For instance, defining Treg cell specificity would allow for a more precise targeting of Treg cell-based therapies to the most appropriate antigens. Many antigens that are targeted by effector cells in autoimmune inflammatory diseases have been defined [e.g., BP180 in bullous pemphigoid (177), desmoglein 3 in pemphigus vulgaris (178), and insulin and other antigens in type-1 diabetes (179)]. Additionally, there is considerable hope that allo-antigen-specific Treg cells will show superior suppressive function compared to polyclonal Treg cells in preventing transplant rejection and graft-versus-host disease. By identifying appropriate antigens, antigen-specific Treg cells could be expanded *ex vivo* and adoptively transferred. Upon interaction with tissue DCs *in vivo*, these Treg cells would likely acquire a tissue-tropic chemokine receptor phenotype and migrate to the same target tissues as their effector T cell counterparts. However, clinical trials with antigen-specific *ex vivo* expanded Treg cells are just starting (180) and it will be crucial to analyze migratory patterns, maintenance in tissues, and suppressive function of these Treg cells.

In addition to controlling their specificity, the identification of tissue- and inflammation-specific Treg cells subsets implies that targeting the "correct" Treg cell population will be critical for effective Treg cell-based immunotherapy. One can envision exposing Treg cells *in vitro* to defined cytokine- and co-stimulatory conditions to induce the expression of specific homing receptors and functional modules with the goal to guide them to the

populations of Treg cells are subject to distinct homeostatic constraints. Central Treg cells (cTr ) access paracrine IL-2 in secondary lymphoid tissues

depends on continued TCR/ICOS signals (middle), and memory Treg cells (mTr ) in the skin are supported by IL-7/IL-7R-mediated survival signals (right).

appropriate target tissue and hone their suppressive mechanisms. These applications of *ex vivo* expanded Treg cells will benefit tremendously from a better understanding of the development of tissue- and inflammation-specific Treg cell populations, and the control of the immunosuppressive mechanisms they employ. The end results of these efforts to better target Treg cells would include not only increased therapeutic efficacy but also a simultaneous decrease in unwanted off-target effects that could be envisioned upon Treg cell transfer (e.g., generalized immunosuppression or increased risk of tumor development).

A major advantage of adoptive Treg cell therapy is its potential for long-lasting effects without the need to persistently treat with immunosuppressive drugs. However, current applications have struggled with instability and loss of Treg cells after transfer. Therefore, identifying the factors that govern Treg maintenance will not only allow for better survival of transferred Treg cells but will also open the door to new therapies aimed at manipulating (both positively and negatively) the abundance of endogenous Treg cells in different tissue sites for treating autoimmunity, promoting transplantation tolerance, enhancing cancer immunotherapy, or resolving chronic infection. Additionally, the identification of key functional mechanisms and molecules that support Treg cell maintenance and function in specific tissue sites will have a tremendous impact on development of immunotherapies. In this regard, a recent study indicating that the surface molecule neuropilin-1 is essential for Treg cell maintenance and function in tumor environments, but not in other tissue sites, is particularly promising for efforts to inhibit Treg cells as an adjunct cancer immunotherapy (181).

The various developmental stages of Treg cells have only just begun to be defined and understood. For most clinically relevant inflammatory settings, it is not known which stage, naïve, central, effector, or memory (or subtypes of these), is most suitable for therapeutic interventions. However, due to their presumed stability and antigen-independent maintenance, memory Treg cells seem ideally suited to mediate long-term immunoregulatory benefits. However, memory Treg cells have only been described in the skin and the uterus and it is an open question whether they can be found in other target organs and whether their requirements for maintenance differ in different tissue sites. Additionally, better defining the developmental relationship between these different Treg cell populations could provide new insights into how to best promote the generation of immunoprotective Treg cells.

The manipulation of Treg cells to alter the outcome of inflammatory responses is the most obvious translational application of our increasing knowledge of Treg cell biology. However, the recent studies indicating that Treg cells can have specialized tissuesupport functions that may lead to a broader range of Treg cell applications. Defining tissue-support functions could yield better therapies for wound healing/tissue-regeneration, metabolic regulation, and potentially other tissue-specific functions. However, more studies on how tissue-resident Treg cells differ from each other and their counterparts found in secondary lymphoid organs are required before we can attempt to therapeutically use and manipulate these populations. Importantly, basic research in this and other key areas of Treg cell biology highlighted in this review will continue in an iterative process with clinical trials, each informing the other as the therapeutic potential of Treg cells is fully realized.

# **ACKNOWLEDGMENTS**

The authors thank J. Michael Stolley for his help with the figures. The authors' work has been supported by grants from the NIH to Iris K. Gratz (AR064554) and Daniel J. Campbell (AR055695, AI067750, AI085130, and HL098067). Iris K. Gratz is also supported by a grant from the Austrian Science Fund (FWF) (J2997- B13) and by the Dystrophic Epidermolysis Bullosa Research Association (DEBRA) International and DEBRA Austria.

#### **REFERENCES**

CD62E-dependent migration but not local proliferation. *J Exp Med* (2003) **197**:751–62. doi:10.1084/jem.20021690

**Conflict of Interest Statement:** 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.

*Received: 15 May 2014; accepted: 30 June 2014; published online: 15 July 2014. Citation: Gratz IK and Campbell DJ (2014) Organ-specific and memory Treg cells: specificity, development, function, and maintenance. Front. Immunol. 5:333. doi: 10.3389/fimmu.2014.00333*

*This article was submitted to Immunological Memory, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Gratz and Campbell. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Impact of inflammatory cytokines on effector and memory CD8 T cells +

# **Marie T. Kim1 and John T. Harty 1,2,3\***

1 Interdisciplinary Program in Immunology, University of Iowa, Iowa City, IA, USA

2 Department of Microbiology, University of Iowa, Iowa City, IA, USA

3 Department of Pathology, University of Iowa, Iowa City, IA, USA

#### **Edited by:**

Kim Klonowski, University of Georgia, USA

#### **Reviewed by:**

Linda S. Cauley, University of Connecticut Health Center, USA Joshua J. Obar, Montana State University, USA

#### **\*Correspondence:**

John T. Harty, Department of Microbiology, University of Iowa, 3-512 Bowen Science Building, 51 Newton Road, Iowa City, IA 52242, USA

e-mail<EMAIL_ADDRESS>
Inflammatory cytokines have long been recognized to produce potent APCs to elicit robust T cell responses for protective immunity. The impact of inflammatory cytokine signaling directly onT cells, however, has only recently been appreciated. Although much remains to be learned, the CD8T cell field has made considerable strides in understanding the effects of inflammatory cytokines throughout the CD8T cell response. Key findings first identified IL-12 and type I interferons as "signal 3" cytokines, emphasizing their importance in generating optimal CD8T cell responses. Separate investigations revealed another inflammatory cytokine, IL-15, to play a critical role in memory CD8 T cell maintenance. These early studies highlighted potential regulators of CD8 T cells, but were unable to provide mechanistic insight into how these inflammatory cytokines enhanced CD8 T cell-mediated immunity. Here, we describe the mechanistic advances that have been made in our lab regarding the role of "signal 3" cytokines and IL-15 in optimizing effector and memory CD8T cell number and function. Furthermore, we assess initial progress on the role of cytokines, such as TGF-β, in generation of recently described resident memory CD8 T cell populations.

**Keywords: signal 3, cytokines, effector, resident memory, memory, CD8 T cells**

# **INTRODUCTION**

Naïve CD8 T cells undergo activation when presented with their cognate antigen following a three-signal model. Professional antigen-presenting cells (APCs) provide the crucial first and second signals through the T cell receptor (TCR) and costimulatory molecules, while innate immune cells contribute inflammatory cytokines to promote optimal accumulation and differentiation of effector CD8 T cells (1). Although the role of inflammatory cytokines in maturing professional APCs to stimulate robust T cell responses has been well described (2), investigation of their direct effect on T cells is ongoing. In the following review, we outline mechanistic studies identified for inflammatory cytokine regulation of various stages of the CD8 T cell response and discuss cutting edge research on the cytokine requirements for generation of the novel resident memory T cell (TRM) population.

# **SIGNAL 3 CYTOKINES AND THE MAGNITUDE OF THE EFFECTOR CD8 T CELL RESPONSE**

Initial studies suggesting that cytokines, particularly IL-12, may be important for signaling directly to T cells were made in *in vitro* cultures of T cells and artificial APCs more than a decade ago. Since then, the importance of IL-12 and type I interferon signaling directly to CD8 T cells for optimal effector cell accumulation has been demonstrated both *in vitro* and *in vivo* (3–7). Despite the clear impact of IL-12 and IFNα/β on effector CD8 T cell numbers, it remained unclear how inflammatory cytokines regulated the magnitude of effector CD8 T cell responses. Although several other cytokines have been discussed in the literature recently for T cell differentiation (8, 9); here, we will focus on signal 3 cytokines as originally defined for their role in T cell accumulation (3–7). After their classification as signal 3 cytokines, IL-12 and type I interferons were proposed to enhance accumulation of CD8 T cells following one of two models: via greater survival (2, 10) or by conferring an early proliferative advantage (11, 12). The model for enhanced survival stemmed from 3 days culture experiments, which demonstrated accumulations of cells in cultures containing IL-12 with no detectable changes in cell division. The latter model was supported also by *in vitro* studies, where IL-12 transiently increased expression of CD25, the high affinity IL-2 receptor, peaking at day 2 (11). Hence, previous reports addressing the mechanism by which signal 3 cytokines allow optimal accumulation of effector CD8 T cells were limited to short-term *in vitro* experiments with no clear answer to the question of whether survival or early proliferation, or both, contribute to the magnitude of the CD8 T cell response. Furthermore, the temporal disconnect between signal 3 cytokine-driven CD25 expression and optimal accumulation of effector CD8 T cells many days later has not been assessed (13). Here, we describe a recent study from our lab addressing these knowledge gaps concerning the mechanism by which signal 3 cytokines allow optimal accumulation of effector CD8 T cells *in vivo*.

Utilizing an OT-I T cell adoptive transfer system followed by DC-OVA priming with or without the TLR9 agonist, CpG, to induce signal 3 cytokines, Starbeck-Miller et al. compared CD8 T cells activated *in vivo* in the presence or absence of signal 3 cytokines (14). Gene expression profiling of T cells from these groups at D7 post immunization clearly showed that signal 3 cytokines enhanced transcription of proliferation, but not

anti-apoptosis-associated genes (14). Additionally, analysis of CD8 T cells primed by DC with or without signal 3 showed no differences in proliferation or total cells numbers as late as day 5 post immunization. Thus, the *in vivo* data do not support either of the proposed models for signal 3 activity. Interestingly, both DC and DC + CpG OT-I cells isolated on D4 and moved into *in vitro* cultures failed to divide, although transfer of the same populations to an *in vivo* host revealed more robust proliferation from the CD8 T cells that had been exposed to signal 3 cytokines. This suggested that signal 3 cytokines established a proliferation program, but sustained proliferation required an additional component that was present in a naïve host. Since IL-2 is an important driver of T cell accumulation, Starbeck-Miller et al. monitored expression of the high affinity IL-2 receptor, CD25, on DC versus DC + CpG CD8 T cells. Indeed, IL-12 and type I interferon sustained CD25 expression, allowing for greater IL-2-induced proliferation via activation of the PI3K pathway and expression of FoxM1, a positive cell cycle gene regulator. Importantly, administering the IL-2 neutralizing antibody JES6 from D4-6 removed the proliferative advantage conferred by signal 3 cytokines. Thus, these studies verify, and add mechanistic insight to the model, indicating that signal 3 cytokines neither enhance survival not provide and early proliferative advantage, but rather sustain expression of the high affinity IL-2 receptor, which extends the duration of proliferation after immunization and permits optimal generation of effector CD8 T cells *in vivo.* Interestingly, the effects of IL-12 and type I interferons are not limited to promoting optimal CD8 T cell accumulation, but offer functional advantages to effector CD8 T cells, such as antigen sensitivity, which will be discussed next.

## **DYNAMIC REGULATION OF ANTIGEN SENSITIVITY BY INFLAMMATORY CYTOKINES**

The protective capacity of CD8 T cells depends on their quantity, functional properties, and anatomical distribution (15). High antigen sensitivity, otherwise referred to as functional avidity, strongly correlates with protective immunity against intracellular pathogens (16). Although T cells cannot directly alter the binding affinity of their TCR through processes like somatic hypermutation, it has been shown that monoclonal TCR-transgenic CD8 T cells can increase their functional avidity from early to late effector time points (17). This study suggested that the functional avidity maturation was a fixed property of CD8 T cells. Here, we describe a mechanistic study demonstrating that inflammatory cytokines directly enhance antigen sensitivity of effector and memory CD8 T cells, however this enhanced sensitivity is not hardwired, but rather tuned by the pathogen-specific milieu.

Using a similar DC immunization protocol as indicated previously, Richer et al. activated OT-I CD8 T cells in the presence or absence of signal 3 cytokines (18). Distinct inflammatory milieu were then initiated by co-infection of DC primed mice with *Listeria monocytogenes* (Lm) or lymphocytic choriomeningitis virus (LCMV) and antigen sensitivity was assessed at day 5 after priming. Strikingly, DC-OVA with LCMV infection substantially enhanced antigen sensitivity by more than 10-fold whereas co-infection with Lm enhanced antigen-sensitivity four to sixfold. To determine whether inflammation increased functional avidity via enhanced TCR signaling, Richer et al. isolated OT-I T cells from DC and DC + LCMV mice on D4 and analyzed phosphorylation of downstream TCR signals after TCR ligation (18). Indeed, inflammatory cytokines dramatically enhanced phosphorylation of ZAP-70, PLCgamma, and ERK1/2 in response to TCR stimulation. Importantly, greater ERK1/2 phosphorylation was not observed with PMA stimulation, which bypasses proximal TCR signals, suggesting that inflammatory cytokines increased the antigen sensitivity of the TCR by enhancing proximal TCR signaling. Consistent with the data from effector CD8 T cells, inflammatory cytokines also increased the antigen sensitivity of memory CD8 T cells by enhancing TCR proximal signaling, albeit to a lesser degree than observed with effector CD8 T cells. This study demonstrated how the pathogen-specific inflammatory milieu affects antigen-sensitivity, an essential functional aspect of both effector and memory CD8 T cells. In addition to signal 3 cytokine effects on memory CD8 T cells, we next review a novel role for IL-15 in memory CD8 T cell trafficking.

# **IL-15-DEPENDENT SYNTHESIS OF SELECTIN LIGANDS**

Numerous studies have described the functional differences between memory and naïve CD8 T cells (13, 19). Among such reports, it was demonstrated that memory, but not naïve, CD8 T cells can be rapidly recruited to inflamed lungs in an antigenindependent manner (20). Importantly, this large influx of memory CD8 T cells was shown to provide immediate cytolytic killing against pathogens expressing cognate antigen (21). Although this non-specific recruitment of memory CD8 T cells was shown to depend on CCR5 expression, the molecular mechanisms initiating early "tethering and rolling" events before chemokine recognition by memory CD8 T cells detection remained undefined.

Immune cell homing is a highly regulated process that begins with selectin family proteins. Leukocytes extravasate into inflamed tissue by constructing ligands to P- and E-selectin, which are expressed on activated endothelium. In contrast, L-selectin mediates homeostatic trafficking of naïve and central memory CD8 T cells through lymph nodes. Previous reports concerning the synthesis of P- and E-selectin ligands had been limited to *in vitro* models, which suggested TCR activation was essential to express appropriate selectin ligands. Herein, we describe studies from Nolz et al. that show P- and E-selectin ligand synthesis occurs on memory, but not naïve, CD8 T cells following inflammation *in vivo* (22). Utilizing the model pathogen, LCMV, Nolz et al. observed uniform expression of functional P- and E-selectin ligands on effector populations, but that most memory CD8 T cells did not express functional P or E-selectin ligands. After detecting high selectin ligand expression on non-specifically recruited memory P14 CD8 T cells following several irrelevant pathogen infections, it was demonstrated, through use of blocking antibodies to P- and E-selectin or P-selectin glycoprotein ligand-1, that non-specific recruitment of memory CD8 T cells to inflamed sites was dependent on selectin binding. To investigate the mechanism regulating inflammation-induced selectin ligand expression on memory CD8 T cells, Nolz et al. analyzed expression of the *Gcnt1* gene, which prompts their formation on naïve, effector, and memory CD8 T cells. Although effector CD8 T

cells expressed high levels of *Gcnt1*, naïve, and memory CD8 T cells had minimal expression of this protein. Interestingly, recombinant IL-15 substantially enhanced P- and E-selectin ligand synthesis on memory, but not naive CD8 T cells *in vitro* and Nolz et al. revealed a similar induction of the Gcnt1 protein via immunoblot. *In vivo*, IL-15-deficiency significantly reduced expression of selectin ligands, and subsequent memory CD8 T cell trafficking to inflamed sites, suggesting that P- and E-selectin ligand expression occurs in an IL-15/STAT5-dependent, but TCRindependent manner. Importantly, IL-15-driven P- and E-selectin ligand expression was shown to occur in human memory CD8 T cells, demonstrating conserved trafficking pathways between mouse and human T cells that can be manipulated for therapeutic purposes.

Until now, IL-15 has been referred to, principally, as a maintenance cytokine for memory CD8 T cells. This study investigating the role of IL-15 in the regulation of core 2 O-glycan synthesis on memory CD8 T cells suggests the possibility of other unexplored functions of this important inflammatory cytokine.

# **TGF-**β**, IL-33, AND TNF REQUIRED FOR RESIDENT MEMORY CD8 T CELLS**

Although the CD8 T cell field has established a paradigm of IL-15 driven homeostatic proliferation as the model of memory CD8 T cell maintenance for circulating T cells, localized CD8 T cell populations in the lung (23), gut (24), and skin (25), among other tissues, have been shown to sustain a sizable pool of memory CD8 T cells despite the absence of IL-15 signaling. Most recently, the TRM population has garnered immense interest for their distinct surface phenotype, local protective capacity, and long-term maintenance in the absence of traditional cytokines. Skin and gut infection models to generate transgenic CD8+ TRM populations are well established (26, 27); hence, we describe recent advances in determining the cytokine signals involved for TRM development and maintenance following either immunization or infection.

Resident memory T cell cells represent a novel, non-circulating class of T cells that persist within extralymphoid tissue and demonstrate superior regional immunity (28). The best-characterized TRM cells express the alpha chain of the αEβ7 integrin (CD103), as well as the sphingosine 1 phosphate receptor (S1PR1) inhibitor CD69, in multiple tissue compartments. Relevantly, both molecules are required for the optimal formation and maintenance of TRM cells in the skin (26). Since *in vitro* and some *in vivo* studies have long since shown that transforming growth factorβ (TGF-β) signaling promotes CD103 expression on immune cells (29–31) and that TGF-β is expressed in the skin epithelium, Mackay et al. investigated whether signaling through the TGFβ receptor was required to upregulate CD103 and establish TRM cells *in vivo* (26, 32). Utilizing one to one adoptive transfer models of WT and *Tgfbr2f/f .*dLck-Cre (Tgfbr2−/−) OT-I T cells into C57BL/6 mice followed by infection with OVA-expressing HSV, Mackay et al. indeed demonstrated that Tgfbr2−/− OT-I cells failed to upregulate CD103 and had a dramatically reduced ability to form TRM.

By utilizing acute and chronic infections with LCMV, Zhang et al. delved further into the mechanism behind TGF-β signaling for generation and maintenance of TRM cells (27). Creating equal ratio mixtures of WT and Tgfbr2−/−P14 T cells followed by either LCMV-Armstrong (acute) or Clone 13 (Cl13, chronic) infections, Zhang et al. notices defective maintenance of Tgfbr2−/− cells in Armstrong, but not Cl13-infected hosts. While monitoring integrin expression in secondary lymphoid organs, which are the major source for TRM cells, Zhang et al. detected enhanced expression of α4β7 on Tgfbr2−/− cells in Cl13, compared to Armstrong-infected mice (27). As α4β7 aids in the migration to the gut (33, 34), it was concluded that, although Tgfbr2−/− TRM cells are locally declining in both Armstrong and Cl13 infected mice, the more prominent, enhanced expression of α4β7 on splenic Tgfbr2−/− T cells of Cl13-infected hosts allowed for continual replacement and stabilization of TRM numbers. Thus, TGF-β acts as a negative regulator to TRM formation through α4β7 downregulation, but is required for the maintenance of established TRM cells in the gut through induction of CD103 expression.

The above findings clearly identify the relationship between TGF-β and CD103 expression for persistence of TRM cells; however, CD103 is not required in all TRM niches (35–37). Thus, we outline a complementary study, defining the transcriptional regulation of a ubiquitous TRM marker, CD69, to establish TRM cells, where CD103 may be dispensable. The antagonistic relationship between CD69 and S1PR1 are well established (38). The zinc-finger transcription factor KLF2 catalyzes the expression of S1PR1, known to promote lymph node egress (39). Hence, Skon et al. initially uses adoptive transfer models of KLF2-GFP P14 T cells followed by LCMV-Armstrong infection to monitor KLF2 expression in circulating, compared to resident memory CD8 T cells (40). As expected, TRM cells expressed low levels of both KLF2 and S1PR1, while CD69 expression was increased. Interestingly, *in vitro* cytokine screening revealed that a combination of TGF-β, IL-33, and TNF were capable of inducing a modest downregulation of KLF2 expression. To analyze the effect of S1PR1 expression on TRM formation, Skon et al. overexpressed S1PR1 through retroviral transduction of P14 cells, and demonstrated that failure to downregulate S1PR1 prevented the establishment of TRM cells in the salivary gland, kidney, lamina propria, and intestinal epithelium (40). Hence, these studies propose that migration to non-lymphoid tissue enhances exposure of CD8 T cells to TGF-β, IL-33, and TNF, which triggers some loss of KLF2 expression, subsequently decreasing S1PR1, and allowing CD69 upregulation. Although the upregulation of CD69 may be controlled by multiple factors, increasing the complexity of this process, these studies, among others, clearly demonstrate that the non-migratory TRM population has novel cytokine requirements for their generation and maintenance (**Figure 1**) and that this list of cytokines may continue to expand.

# **SYNOPSIS**

In this review, we outline recent studies uncovering the mechanisms by which inflammatory cytokines regulate various attributes of circulating and resident memory CD8 T cell populations. Although investigation of the role of inflammatory cytokines on TRM cells, and T cells in general, remain far from complete, the field

**FIGURE 1 | Cytokines involved in TRM formation and maintenance**. Early after infection, local TGF-β signals prevent migration of effector CD8 T cells from the spleen to non-lymphoid tissue by downregulating the αEb7 integrin. However, tissue-specific programming during priming of CD8 T cells causes homing to appropriate resident tissue. In addition, the transcription factor KLF2 gets downregulated as effector CD8 T cells travel to non-lymphoid tissue toward a combination of TGF-β, IL-33, and TNFα signals, which causes a decrease in S1P1, allowing upregulation of CD69. Within resident tissue, TGF-β signals sustain TRM numbers.

has made remarkable progress in understanding how the inflammatory environment can directly modulate the number, function, migration, and maintenance of T cells.

# **ACKNOWLEDGMENTS**

Dedicated to our friend, colleague, and competitor Leo Lefrançois. We would like to acknowledge members of the Harty lab for helpful discussion.

#### **REFERENCES**

CD16- NK cells with similarities to decidual NK cells. *Proc Natl Acad Sci USA* (2007) **104**(9):3378–83. doi:10.1073/pnas.0611098104

**Conflict of Interest Statement:** 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.

*Received: 03 April 2014; paper pending published: 12 May 2014; accepted: 05 June 2014; published online: 19 June 2014.*

*Citation: Kim MT and Harty JT (2014) Impact of inflammatory cytokines on effector and memory CD8*+ *T cells. Front. Immunol. 5:295. doi: 10.3389/fimmu.2014.00295 This article was submitted to Immunological Memory, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Kim and Harty. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Blowing on embers: commensal microbiota and our immune system

# **Darina S. Spasova1,2 and Charles D. Surh2,3,4\***

1 Kellog School of Science and Technology Doctoral Program in Chemical and Biological Sciences and the Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA

2 Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA

3 Academy of Immunology and Microbiology, Institute of Basic Science, Pohang, South Korea

4 Department of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, South Korea

#### **Edited by:**

Kimberly Sue Schluns, University of Texas MD Anderson Cancer Center, USA

#### **Reviewed by:**

Joshua J. Obar, Montana State University, USA Peter John Lane, Birmingham University, UK

#### **\*Correspondence:**

Charles D. Surh, Academy of Immunology and Microbiology, Institute of Basic Science, Room 205 POSTECH Biotech Center, San 31, Hyoja-dong, Pohang 790-784, South Korea e-mail<EMAIL_ADDRESS>
Vertebrates have co-evolved with microorganisms resulting in a symbiotic relationship, which plays an important role in health and disease. Skin and mucosal surfaces are colonized with a diverse population of commensal microbiota, over 1000 species, outnumbering the host cells by 10-fold. In the past 40 years, studies have built on the idea that commensal microbiota is in constant contact with the host immune system and thus influence immune function. Recent studies, focusing on mutualism in the gut, have shown that commensal microbiota seems to play a critical role in the development and homeostasis of the host immune system. In particular, the gut microbiota appears to direct the organization and maturation of lymphoid tissues and acts both locally and systemically to regulate the recruitment, differentiation, and function of innate and adaptive immune cells. While the pace of research in the area of the mucosal–immune interface has certainly intensified over the last 10 years, we are still in the early days of this field. Illuminating the mechanisms of how gut microbes shape host immunity will enhance our understanding of the causes of immune-mediated pathologies and improve the design of next-generation vaccines. This review discusses the recent advances in this field, focusing on the close relationship between the adaptive immune system and commensal microbiota, a constant and abundant source of foreign antigens.

**Keywords: commensal, microbiota, immune system,T cells, regulation**

# **INTRODUCTION**

Microorganisms represent the oldest and most ubiquitous forms of life on our planet. These microbes evolved alongside other organisms, like metazoans, with a portion of them colonizing and establishing life-long relationships with larger species. Such mutualistic relationships have been described in plants (1), insects (2), worms (3, 4), fish (5, 6), birds (6), and mammals (7). Shortly after birth, the mammalian mucosal tissues are exposed to the environment and colonized by viruses, bacteria, protozoa, and fungi, resulting in the formation of complex populations of microbes, collectively called the commensal microbiota. The mammalian commensal microbiota constitutes of over 1000 different species of microbes and outnumbers the host cells by 10-fold (8, 9). The intestinal mucosa harbors the largest amount of microbes in the human body; many are beneficial while some, termed pathobionts, are generally harmless, but can turn pathogenic with an imbalance of the microbial community. The commensal microbiota has long been appreciated for its essential contributions to host development and health. Such benefits include metabolism of indigestible food byproducts, generation of nutrients, defense against pathobionts, promotion of angiogenesis and enteric nerve function, maintenance of intestinal structure, and contribution to the development and regulation of the mammalian immune system (10–15).

For better and for worse, the mammalian immune system is one aspect of host physiology that is heavily influenced by commensal microbiota. Both human and animal model research supports the hypothesis that immune-related disorders like inflammatory bowel disease (IBD) (16), cancer (17), diabetes (18, 19), allergies (20), and even obesity (21, 22) may result from dysbiosis of the commensal microbial communities. To better understand how microbiota contributes to the onset and/or exacerbation of such disorders (23, 24), it is important to understand how signals from intestinal microbiota influence the immune system under normal and diseased conditions. This review discusses the effect of gut microbiota on the mammalian immune system, with a particular focus on T cell differentiation, responses, and homeostasis. First, we discuss how the host senses microbes. Then, we review the ability of dendritic cells (DCs) to induce immunogenic or tolerogenic responses to microbial signals. Finally, we examine the signals that microbes provide and their role in modulating T cell differentiation and function.

# **HOST SENSING OF MICROBES**

Commensal microbiota may be largely harmless but it is nevertheless crucial to maintain barriers to prevent opportunistic invasions and, occasionally, an immune response is required to keep potential pathobionts in check. In mucosal tissues, such a response is promoted by both epithelial and innate immune cells via ligation of various receptors expressed differentially in these cells. The recognition of microbial particles such as DNA, cell wall components, and metabolites occurs in the context of innate toll-like receptors (TLRs), NOD-like receptors (NLRs), G protein-coupled receptors (GPCRs), other pattern-recognition receptors (C-type lectin receptors, RIG-I-like receptors, and AIM-2-like receptors), and yet to be identified receptors expressed by both hematopoietic and non-hematopoietic lineage cells in mucosal tissues. The following section will focus on the use of TLRs and NLRs by the host to sense microbes.

#### **TOLL-LIKE RECEPTORS**

Toll-like receptors are pattern-recognition receptors, which bind to evolutionarily conserved molecules expressed by bacteria, viruses, and other microorganisms (25). Some of them (TLRs 2, 3, 4, and 5) are expressed on the cell surface, whereas others (TLRs 3, 7, and 9) are expressed in endosomal compartments of cells (26, 27). TLRs are expressed in high levels on epithelial cells and innate immune cells and can sense various microbial molecules such as double- and single-stranded RNA, LPS, flagellin, CpG (28), ensuring detection of all microbes.

Toll-like receptor ligands have been implicated in the onset and management of various diseases. For instance, TLR signaling has been shown to affect Crohn's disease and psoriatic arthritis in humans (29, 30). Studies from the non-obese diabetic (NOD) murine model of type-1 diabetes have also revealed that disruption of TLR signaling protects these mice from developing diabetes (31). These data indicate that microbial recognition via TLRs has a significant implication in the onset of autoimmune and inflammatory diseases.

Toll-like receptor ligands have also been suggested to modulate mucosal cell function and tissue maintenance. One mechanism is by inducing the synthesis of antimicrobial peptides and tissuerepair factors by intestinal epithelial cells (IECs) (32). Moreover, LPS-induced TLR signaling has been linked to NFκB activation of IECs, which results in promoting either a tolerogenic or a proinflammatory environment depending on the microbial context (33). LPS also acts directly on mucosal DCs, affecting their activation and migration to various anatomical sites where they can induce adaptive responses (33). Similar to IECs, TLR signals can induce either activation or tolerance programs in DCs (discussed below).

#### **NOD-LIKE RECEPTORS**

NOD-like receptors contain multiple subfamilies and are expressed by many organisms from plants to mammals. Mammalian NLRs play a crucial role in sensing microbes and inducing various pro-inflammatory programs in response to microbial products (34). NLRs are not only essential in responses to pathogens but they are also necessary for the development of intestinal lymphoid tissues, maintenance of commensal communities, and mounting antigen-specific immune responses (35). This section will discuss only the NLRC and NLRP subfamilies of NLR signaling. For a more comprehensive discussion of NLRs, see the recent review by Chen et al. (36).

The NLRC subfamily contains NOD1, which recognizes peptidoglycan-containing mesodiaminopimelic acid (iE-DAP), and NOD2, which recognizes muramyl dipeptide (MDP) (37). One of the major functions of NOD1/2 is the activation of NFκB signaling. Pathogen induction of NOD2 results in NFκB activation and secretion of pro-inflammatory cytokines, especially IL-1 (34). Mutations in NOD2 are linked to Crohn's disease; here, the inability to induce NFκB signaling on IECs and immune cells is thought to impair intestinal mucosal barrier function, resulting in pathologies from invasion of the intestine by commensal bacteria (38). Support for this notion was obtained from work in the mouse. Hence, increased intestinal permeability to commensal bacteria was reported in NOD2 deficient mice, indicating that NOD2 signaling is important for maintaining intestinal epithelial integrity and barrier function (39). Similarly, NOD1 also has a role in maintaining intestinal homeostasis but in other related ways. Thus, NOD1 deficient mice possess a decreased number of intestinal lymphoid follicles and lower expression of CCL20 as well as a change in microbiota composition (35). NOD1 and 2 signaling has been implicated in both pro-inflammatory and immunoregulatory roles as it can induce secretion of IFN-γ, IL-12, IL-6 but also of IL-10 in various models of disease, demonstrating thus suggesting that a fine balance of NLRC signaling must be maintained (40–43). It is clear that NOD1 and NOD2 contribute to regulatory and inflammatory responses to microbes but it is the integration of these signals and those from other receptors, such as NLRP, that likely result in determining whether a regulatory or inflammatory response is appropriate.

NLRP and NLRC signaling are closely associated with the immune system as they affect inflammasome function. The inflammasome is a multiprotein complex that is required for the enzymatic processing of pro-forms of IL-1β and IL-18. Multiple NLRs can activate the inflammasome but they all recognize bacterial flagellin or type III secretion system (44). It has been suggested that NLRP and NLRC signaling is more specific than NOD1/2 signaling since it is triggered not only by these two types of bacterial molecules but it also has regulatory molecules in place that are recruited only by certain species of bacteria that merit an inflammatory response (44, 45). A recent model proposes that inflammasome activation to various microbes is mediated commonly by NLRC4, but the specificity comes from differential NLRP recognition of products from specific bacterial species (46). NLRP4, NLRP3, and NLRP1 have been associated with induction of the inflammasome in response to bacterial, fungal, and viral products; mutations in these genes are associated with higher susceptibility to bacterial and viral infections as well as autoimmunity (47–49).

Even though NLRP activation of the inflammasome was believed to be largely in response to infection and severe cellular stress, it was recently demonstrated that NLRP6 plays a crucial role in regulating interactions with commensal microbiota during steady-state conditions. NLRP6 knockout animals were highly susceptible to DSS-induced colitis and had decreased intestinal IL-18 levels as well as altered microbiota composition (50). It is unclear exactly what triggers NLRP6-mediated activation of the inflammasome but it likely occurs in response to a collection of environmental signals. Because NLRP activation can occur in the context of microbiota, stress, or infection, the discrimination between pathogenic and commensal microbes likely involves the integration of multiple signals from the inflammasome, NFκB signaling, and other pathways by innate immune cells.

# **INNATE LYMPHOID CELLS**

Innate lymphoid cells (ILCs) comprise a recently described population of innate immune cells, which have been shown to regulate immunity, inflammation, and tissue repair in various anatomical sites (51–53). Like B and T cells, they differentiate from a common lymphoid progenitor but, unlike B and T cells, they lack a rearranged antigen-specific receptor (51–53). Nonetheless, ILCs closely resemble CD4 helper T cells and express many of their transcription factors and molecules. Multiple subsets of ILCs exist, and they are classified into three groups – Groups 1, 2, and 3 – according to the expression of distinct transcription factors and effector molecules (51–53). Group 1 ILCs, such as NK cells, are characterized by the expression of the transcription factors T-bet and/or Eomesodermin and the production of the cytokine IFN-γ in response to stimulation with IL-12 and IL-18 (51–53). Group 2 ILCs' canonical features are the expression of the transcription factors RORα and GATA3 and the expression of IL-5 and IL-13 in response to stimulation with IL-25, IL-33, and TLSP (51–53). Finally, group 3 ILCs, such as lymphoid tissue inducer (LTI) cells, NKp46+, and NKp46− cells, are characterized by the expression of RORγt and/or T-bet and/or the aryl hydrocarbon receptor (AHR) and upon stimulation with IL-23 and IL-1β, they produce IL-22 and/or IL-17 and/or IFN-γ (51–53).

Innate lymphoid cells inhabit various tissues where they have been shown to direct inflammation at mucosal surfaces, especially in a response to infection, and confer resistance to pathogens (54). Group 1 ILCs include NK cells and a population of human mucosal ILCs, which lack expression of NK surface markers but produce high levels of IFN-γ in response to IL-18 and IL-12 stimulation (55). They are present in high levels in patients with Crohn's disease and contribute to increased inflammation in the intestine (56). A new NKp44+CD103+ population of ILC1s, which produces large amounts of IFN-γ, CCL4, and TNFα, has been recently described in the human intestine (57), and it has been suggested that it is the innate counterpart of tissue-resident memory CD8 T cells. Group 2 ILCs participate in protection against helminth infection in the intestine by producing IL-25 (58–60) as well as in wound healing in the airways via the production of amphiregulin, which induces epithelial cell proliferation (61, 62). Group 3 ILCs have been shown to regulate CD4 T cell responses to commensal bacteria in the intestine of mice as well as to prevent bacterial dispersal to peripheral tissues (63, 64). Recently, it has also been revealed that intestinal macrophages act through ILC3 to induce the production of tolerogenic molecules (IL-10 and retinoic acid) by intestinal DCs or macrophages and thus promote the induction and homeostasis of regulatory T cells in the intestine and the mesenteric lymph nodes (65). Even though ILCs are a novel population of innate immune cells, it has been well demonstrated that they play a key role in mucosal defense from pathogens as well as in maintenance of mucosal tissues.

# **DENDRITIC CELL FUNCTION DEPENDS ON LOCATION AND ENVIRONMENT**

Mucosal DCs are in constant contact with commensal microbiota via various pattern-recognition receptors. Because DCs have the unique ability to elicit a robust T cell response (66), they are differentially conditioned based on anatomical location and local antigenic load to be either tolerogenic or pro-inflammatory in response to the same signal. In the lungs, TLR4 signaling on DCs results in antigen-specific CD4 T cell mediated inflammation (67). On the contrary, TLR4 signaling in intestinal DCs is shown to be tolerogenic (68), suggesting that the environmental cues and location determine how a DC integrates such signals. In the context of other TLR ligands, administration of CpG in TLR4 deficient mice reduces their susceptibility to systemic allergy development, arguing for a tolerogenic role of TLR9 signaling (69). In contrast, when CpG is administered to germ-free mice, it causes increased IFN-γ and IL-17 production in the intestine of these mice, conferring protection against intestinal parasites, indicating a more activating role for TLR9 (69). Based on these data, it is widely accepted that the environmental context in which DCs integrate the same signal determines whether an inflammatory immune response occurs.

At mucosal sites, unlike in other lymphoid tissues, DCs perform unique roles and occupy distinct niches (70). In addition to specifying the inflammatory or anti-inflammatory nature of the T cell response, DCs also direct the homing of effector cells. In the mesenteric lymph node, they can promote upregulation of CCR9 and α4β7 on B and T cells (71, 72), thereby facilitating their migration back to the intestine (73, 74). In the intestine, DCs translocate to and reside in the lamina propria (LP), Peyer's patches, isolated lymphoid follicles, and mesenteric lymph node, where they sample and present luminal and self antigens to T cells (75). In the Peyer's patches, DCs are divided into three groups: CXCR1+ DCs, CCR6+ DCs, and CCR7+ DCs. Each group has distinct functions and characteristics. CXCR1+ DCs are found in the Peyer's patch in close proximity to M cells, positioned there with the purpose to sample luminal antigens in a TLR-dependent manner (76). CCR6+ DCs are migratory and are found in the dome of the Peyer's patches, from where they readily translocate to the follicle-associated epithelium in response to microbial stimulation. Finally, CCR7+ DCs are found in the T cell areas of the Peyer's patches, where they can induce T cell activation (77, 78) and migration (73, 74), and IgA production by B cells (79, 80) in response to microbial signals (81).

As Peyer's patches are rare along the intestinal tract, the majority of gut-associated DCs are more frequently found in the LP of the small intestine. LP DCs, often differentiated from circulating precursors (82), express tight junction proteins that allow them to reach their dendrites between IECs and sample the luminal contents directly (83). This phenomenon depends on CX3C chemokines and TLR ligation (84, 85). CX3CR1 deficient mice exhibit impaired luminal sampling by DCs and are thus more susceptible to *Salmonela typhimurium* infection (85). This finding suggests that luminal sampling by DCs may be critical for protective immune responses against intestinal pathogens.

As mentioned above, intestinal DCs are thought to be more tolerogenic than systemic DCs (86, 87). It has been shown that stimulation of intestinal DCs with LPS results in elevated IL-10 secretion, whereas the same stimulation of circulating DCs results in the production of a variety of pro-inflammatory cytokines (88, 89). The mechanisms that condition intestinal DCs to produce IL-10 in response to LPS stimulation, but not systemic DCs, are still largely unknown. It has been suggested that reduced TLR expression (88, 89) and hyporesponsiveness to TLR ligation (68) as well as negative regulation of NFκB pathways via NOD2 signaling play a role in the desensitization of intestinal DCs to microbial antigens (40–43).

Dendritic cells play a key role in promoting T cell differentiation and responses to microbiota in the gut as well as systemically. Some studies have shown that intestinal DCs can transport self and microbial antigens to the mesenteric lymph nodes (90) where they orchestrate T cell activation and differentiation into effector cells. Intestinal DCs are not exclusively tolerogenic and can induce pro-inflammatory T cell responses by secreting IL-12 and IL-23. IL-12 is a master inducer of T helper 1 (Th1) responses (91) and thus plays an important role in Th1-associated autoimmune diseases, such as IBD (92–94). IL-23 has been implicated in inflammatory T helper 17 (Th17) responses (95) in murine models of joint (96) and intestinal inflammation (97–100) as well as psoriasis (101, 102). Conversely, in addition to Th1 and Th17 responses, intestinal DCs can induce T regulatory cells in the context of retinoic acid, TGF-β, and IL-10 in the gut, suggesting that intestinal DCs can serve to direct T cell differentiation so as to induce the appropriate response based on different contexts (75, 86, 87). DCs can sense microbial antigens from commensal and pathogenic microbes and ideally are able to differentiate between the two in order to induce appropriate T cell responses, protecting the host from infection but at the same time creating a tolerogenic environment for the commensal microbiota to thrive.

# **COMMENSAL MICROBIOTA MAINTAINS T CELLS' POISED STATE**

Many dramatic effects of commensal microbiota on host T cells have been documented. Following microbial colonization, immune cells are recruited, induced to differentiate and to reside in the gut (13). At steady state, the intestine houses a large number of T cells, which produce IL-17, IL-22, IFN-γ, and IL-10 (69, 103). In the absence of microbiota, there are deficiencies in the production of these cytokines (15, 103), indicating that commensal microbes control their constitutive production in the gut. CD4 T cell numbers have been shown to be decreased in germ-free mice, affecting T helper 1 (Th1) and T helper 17 (Th17) cells, although regulatory T cell (Treg) frequencies remain the same (69, 103). When germ-free mice are conventionalized, a broad Th1, Th17, and Treg expansion occurs and cytokine production is recovered in the gut, indicating that the accumulation of functional CD4 T cells in the gut is microbiota dependent (104). Furthermore, upon infection with a mucosal pathogen, CD4 T cells in the intestine respond to both the pathogen as well as to the commensal microbiota that permeate the intestinal epithelial layer, demonstrating the ability of the adaptive immune system to overcome its tolerance to commensals and its ability to contain pathobionts and opportunistic pathogens (105). Among the CD8 T cell populations in the gut, intraepithelial lymphocytes (IELs) are significantly decreased in

germ-free mice, and they are restored upon colonization (106). Because the host needs metabolites and essential nutrients from microbes, yet at the same time it must protect itself from infection, maintaining a balance between pro- and anti-inflammatory T cell populations in the gut is essential.

In addition to affecting the local populations, there is evidence supporting the notion that commensal microbes also influence the generation and function of the host immune system more broadly. Peripheral lymphoid organ structure and function are disrupted in the absence of commensal microbiota (107). Moreover, T cell responses to systemic antigens are also altered in the absence of commensal microbiota. Recent studies have suggested that commensal microbiota keeps the immune system primed and ready to respond during the steady state and conventional mice respond more robustly to infection than germ-free mice (108). This section will discuss the effect of commensal microbiota on T cell responses both at mucosal sites and systemically.

#### **TH17 CELLS**

IL-17 producing Th17 cells are represented in high numbers in the LP of the small intestine, where they play a role in protection against extracellular pathogens (103). The differentiation of Th17 cells is dependent on their expression of the transcription factor RORγt, and it is driven by signals from TGF-β and IL-21 or IL-6 (109). Additionally, Th17 cells require IL-23 for maturation and survival (110). As the number of Th17 cells is dramatically decreased in the small intestine of germ-free animals, there is a widely accepted notion that commensal bacteria are required to cue Th17 differentiation (103). A TLR-independent mechanism for the promotion of Th17 differentiation in the gut has been suggested, as signals through MyD88 are not necessary for the induction of Th17 cells (103). Furthermore, data from mice mono-associated with segmented filamentous bacteria (SFB) indicate that even a single species of commensal bacteria is enough to direct T cell differentiation toward a Th17 bias (104, 111).

Not only do microbiota-derived signals stimulate the differentiation and accumulation of Th17 cells in the intestinal LP, but they also maintain Th17 cell homeostasis and survival. Studies have indicated that treatment with the antibiotic vancomycin decreases Th17 populations in the small intestine of conventional mice (103, 112), whereas treatment with a complex cocktail of antibiotics, results in a decreased Th17 frequency in the mesenteric lymph node (113). In other mucosal sites, such as the skin, resident commensal microbiota have been shown to induce Th17 and Th1 differentiation to protect the host from pathogens and possible opportunistic microbes (114).

#### **REGULATORY T CELLS**

Maintaining tolerance to resident bacteria is the key to preventing inflammatory diseases in mucosal tissues. Before the importance of tolerance was widely accepted, it was commonly believed that an unknown pathogen was the trigger for IBD. However, successful treatment of the disease with immunosuppressive drugs has lead to the hypothesis that IBD may result from defects in tolerance to otherwise non-pathogenic gut commensals (115–118).

The idea that T cells must be tolerized to commensal microbiota was suggested decades ago, when in an adoptive transfer model, naïve CD4 T cells caused colitis (119) but were held in check by another population of CD4 T cells (118), now known as regulatory T cells (Tregs). For the purposes of this review, Tregs are T cells that express the master regulator transcription factor, Foxp3, and display anti-inflammatory activity, including secretion of TGF-β and/or IL-10 (120). Extrapolation from early studies led to the proposal that regulatory T cells are required to prevent aberrant T cell responses to resident microbes. Scientists were thus surprised when data from the small intestine of germ-free mice emerged to show that commensal bacteria are not necessary for the development of Tregs in that organ (121–123). Furthermore, when germ-free Tregs were assayed, they were able to suppress colitis-like symptoms albeit not as well as conventional Tregs in the adoptive transfer model of the disease (122, 124), indicating that functionally and developmentally, small intestinal Tregs are independent of commensal microbiota. Even though the small intestine houses a large amount of T cells, it is the colon that houses the largest load of microbiota and is the site of colitis observed in these experiments.

Recent studies on the effect of gut microbiota on Treg development and function have elucidated that colonic Tregs are significantly decreased in germ-free mice, indicating that, in the colon, commensal microbiota is the major inducer of colonic Tregs (125, 126). The exact mechanism of how the induction of colonic Tregs occurs remains unknown. It has been suggested that certain species of microbiota, such as Clostridium clusters IV and XIVa, can induce Foxp3+ Treg generation in the colon (125). In order to determine whether commensal microbiota is required for the differentiation of naïve peripheral T cells into colonic Tregs, several groups looked at the expression of an Ikaros-family transcription factor, Helios, which is thought to signify thymic origin of Tregs. They showed that in the colons of germ-free mice, most of the Tregs were Helioshi, suggesting that these T cells became Tregs in the thymus (so-called thymic or tTreg), whereas their counterparts in conventional mice were mostly Helioslo, indicating that these CD4 T cells converted to Tregs in the peripheral tissues (so-called peripheral or pTreg) (125, 127). These results suggested that in conventional hosts, colonic Tregs are differentiated outside of the thymus (presumably in the gut) in response to foreign antigens. Because the use of Helios in the field is still quite controversial (128, 129), other experimental approaches were utilized to confirm the importance of commensal microbiota on colonic Treg differentiation and function. Subsequent work using Neuropilin-1 (Nrp-1) as a marker to detect tTregs confirmed that induction of pTregs in the colon was mediated by commensal microbiota (130). Another study using a fixed TCRβ showed that colonic Tregs utilize different TCRs from systemic circulating Tregs, reaffirming the notion that they recognize distinct gut antigens (127). Additionally, TCRs from colonic Tregs, expressed as transgenes, were unable to induce generation of tTregs, again supporting the notion that colonic Tregs were peripherally induced in response to commensal antigens (127).

As the mechanism is still unknown, multiple models have been offered to explain how microbiota supports an abundance of pTregs in the gut. It has been proposed that preferential expansion of pTregs occurs in the intestine either by microbial components influencing pTregs directly or indirectly through the products of bacterial metabolism, such as short-chain fatty acids (131– 133), and from presentation of bacterial peptides (such as PSA) by innate immune cells (134–136) and/or from effector T cells (129, 137, 138). Another hypothesis offers that the foreign antigen load in the gut is so large that, given a limited capacity for antigen presentation, self-peptides are displaced and the increased prevalence of pTregs simply reflects the increased density of their targets (i.e., foreign-peptide/MHC-II) and decreased availability of tTreg targets (i.e., self-peptide/MHC-II) (127). The question of the specificity of small intestinal pTregs still remains open. It is possible that these Tregs are generated in response to self or dietary antigens, as the abundance of these antigens is greater in the small intestine than in the colon.

Fostering a balance between tolerating commensal microbiota and maintaining the ability to mount an immune response to microbial pathogens is crucial for the survival of the host. Despite the seeming bias toward induction of tolerogenic responses in the gut, immune responses still occur readily against pathogens to protect the host from infections. The mechanisms utilized by the host to distinguish between the commensal and pathogenic bacteria are still poorly understood and are under intense investigation.

### **INTRAEPITHELIAL LYMPHOCYTES**

Because the intestinal mucosa harbors various opportunistic bacteria, the host has evolved the ability to house cytotoxic killer T cells in close proximity to potential sites of pathogen entry, like the intraepithelial layer of the intestine. The lymphocytes that reside there, also known as IELs, are composed of CD8 T cells that are recruited to and remain in that compartment for the duration of the host's life. There are three types of IELs, each bearing different characteristics and functions: αβ TCR CD8αα, αβ TCR CD8αβ T cells, and γδ TCR T cells. Whereas αβ TCR IELs respond mainly to pathogenic challenge of the epithelial mucosa, γδ TCR IELs participate in would healing and tissue repair by producing pro-inflammatory cytokines and chemokines and recruiting neutrophils, eosinophils, and T cells.

αβ TCR IEL differentiation and maintenance depend on TLR recognition of bacterial signals as MyD88 deficient and germ-free mice exhibit diminished numbers of these cells (139–141). Functionally, in germ-free and antibiotic-treated mice, the cytotoxic activity of αβ TCR IELs is significantly decreased when compared to conventional mice (140), a phenotype that can be rescued by supplying exogenous endotoxin. This suggests that αβ TCR IEL function is both induced and maintained by ubiquitous bacterial components (139, 142).

Similarly, recent data have elucidated the dynamic relationship between commensal bacteria and γδ IELs during mucosal injury. γδ TCR IELs restrict the spread of bacteria to the mesenteric lymph node following intestinal injury as shown by the lack of such a response in γδ TCR IEL-deficient mice (143). γδ TCR IELs exhibit their functions by producing keratinocyte growth factor, which causes epithelial cell proliferation and restoration of barrier functions in the gut (144). Germ-free animals, even though they have a similar numbers of γδ IELs as their conventional counterparts, have significantly decreased ability to promote mucosal injury repair and prevention of invasion by opportunistic pathogens (143). This problem is evidenced by their decreased ability to produce antimicrobiotal peptides, such as RegIIIγ, as well as pro-inflammatory chemokines and cytokines, such as IL-1β, KC, and MIP2α (143), indicating that commensal microbes are required to promote γδ IEL function.

Continued research will reveal the crosstalk between microbiota and these two populations of IELs as they directly relate to intestinal homeostasis. IELs are able to produce both anti- and pro-inflammatory cytokines and promote both strong cytotoxic and tissue-repair responses.

#### **SYSTEMIC T CELL RESPONSES TO INFECTION**

Effects of gut microbiota on innate immune responses to systemic viruses and bacteria have been well demonstrated in the literature (108, 145–147). However, the mechanisms whereby microbiota affects non-mucosal T cell responses have been difficult to comprehend as peripheral T cells are not in direct contact with commensal bacteria. Because T cells confer long-term protection and memory against pathogens, understanding the role of microbiota on T cell responses to infection is crucial. Studies from the 1970s indicated that germ-free and conventional animals exhibit similar immune responses to systemic infection by *Salmonela paratyphi* or lymphocytic choriomeningitis virus (LCMV) (146).

A few groups infected germ-free mice with systemic pathogens in the 1990s. Even though they compared mainly antibody production to assess the immune responses in these mice, a speculation can be made that antibody production is directly connected to T cell responses as T cell help is required for class switching and somatic hypermutation. One group, which infected mice with MCMV and subsequently with *Klebsiella pneumoniae,* concluded that germ-free mice were significantly more susceptible to both pathogens and did not clear the infection in various organs at the same rate as SPF counterparts (148). Moreover, they showed increased mortality by bacterial infection (148). Histologically, the spleens and livers of germ-free animals were more severely affected by the infection and recovered more slowly (148). Another group, which infected germ-free mice with *Salmonella typhimurium,* showed that they were more susceptible to systemic infection than conventional controls (149). Furthermore, they noted a decrease in IgG and IgM responses in the germ-free mice. It could be inferred that the inability to clear a viral and intracellular bacterial infections as well as the decreased antibody production is a result of an impaired T cell response in germ-free mice. Indeed, with improved techniques, more recent work has suggested that systemic immune responses are dampened in the absence of commensal microbes.

Germ-free animals were found to have a higher susceptibility to a prolonged and non-limiting *Listeria monocytogenes* infection compared to conventional animals, a phenomenon supposedly due to the inability of germ-free mice to accumulate T cells at inflammation sites (145, 150). Although *Listeria monocytogenes* is naturally an intestinal pathogen, the method utilized in these studies resulted primarily in infection of the host spleen. Likewise,adaptive immune responses to viral infection of non-mucosal sites are also shaped by the presence of commensal microbes. Antibiotic-treated mice generate significantly fewer virus-specific effector CD4 and CD8 T cells when compared to untreated controls during influenza infection (151). Functionally, CD4 and CD8 T cells in antibiotic-treated mice produce lower amounts of pro-inflammatory cytokines, which correlates with increased virus titers in these mice (151). Further investigation by Abt et al. indicated that antibiotic-treated mice more readily succumbed to influenza infection when compared to conventional counterparts (108). T cell responses in antibiotic-treated mice indicated a decrease in CD8 virus-specific T cells, further confirming that commensal microbiota modulates T cell responses to systemic viral infection (108). The authors concluded that the higher susceptibility of antibiotic-treated mice to infection was due to an inability of macrophages to respond to type-1 interferon and limit viral replication. However, they did not expound upon the relationship between the macrophage defect and the CD8 T cell phenotype they observed. It could be inferred that commensalderived signals provide tonic signaling to innate immune cells, which in turn, influence the ability of these cells to effectively activate naïve T cells and convert them to fully functional effectors. A detailed understanding on the effect of commensal microbiota on systemic T cell responses is yet to be provided.

# **CONCLUSION**

Commensal microbiota plays a crucial role in the development, homeostasis, and regulation of the immune system. With the current rise of autoimmune and inflammatory diseases,the importance of inducing and maintaining tolerance to commensal bacteria is increasingly appreciated. As a constant source of foreign antigens, microbiota plays a pivotal role in inducing tolerance to beneficial bacteria as well as in maintaining the immune system poised to defend the host against pathogens. Since the adaptive immune system is often implicated in microbiota-associated inflammatory and autoimmune conditions, understanding its relationship with commensals is crucial. Metaphorically speaking, if the host immune system is a house,the commensal microbes may be likened to the embers in a fireplace. At homeostasis, they remain glowing, providing constant minimal heating for the house. However, when the house so requires, i.e., when an immune response is necessary, they help create a powerful flame.

#### **ACKNOWLEDGMENTS**

We would like to thank Christopher E. Martin and Bryan R. Becklund for critical reading of this manuscript.

#### **REFERENCES**

regulation of tissue-selective T cell homing. *J Exp Med* (2005) **202**:1063–73. doi:10.1084/jem.20051100

**Conflict of Interest Statement:** 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.

*Received: 22 May 2014; accepted: 24 June 2014; published online: 28 July 2014. Citation: Spasova DS and Surh CD (2014) Blowing on embers: commensal microbiota and our immune system. Front. Immunol. 5:318. doi: 10.3389/fimmu.2014.00318 This article was submitted to Immunological Memory, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Spasova and Surh. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Resident-memory CD8T cells and mTOR: generation, protection, and clinical importance

# **Ryan T. Sowell and Amanda L. Marzo\***

Department of Immunology and Microbiology, Rush University Medical Center, Chicago, IL, USA \*Correspondence<EMAIL_ADDRESS>
#### **Edited by:**

Kimberly Sue Schluns, University of Texas MD Anderson Cancer Center, USA

#### **Reviewed by:**

Jonathan Powell, Johns Hopkins University, USA Xiao-Ping Zhong, Duke University Medical Center, USA

**Keywords: memory, CD8 T cells, tissue resident, mTOR, mucosa**

Tissues such as the lung, skin, intestinal epithelium, and reproductive tract serve as a barricade against pathogen exposure for the entire body. Specifically within the skin and mucosal tissues, a population of resident CD8 T cells plays a salient role in the protection against infection. Resident-memory CD8 T cells (TRM) are a long-lived subset of memory CD8 T cells that do not re-circulate after taking up residence in the tissues. Traditionally memory CD8 T cells were conceptually divided into two subsets central (TCM) and effector (TEM) memory where TCM preferentially localized within secondary lymphoid tissues (SLO) and TEM circulated throughout the peripheral tissues. While the concept of TCM and TEM has been considerably explored, memory CD8 T cells found within barrier tissues do not totally fit within the TCM/TEM paradigm. Through the study of circulating CD8 T cells, our understanding of memory CD8 T cells has grown tremendously in the last 25 years. We now understand that it is not sufficient to simply generate large numbers of circulating memory CD8 T cells in order to enhance protection against localized infection. Developing clinical strategies that can enhance protection against mucosal pathogens will require a clear understanding of how memory CD8 T cells are generated and maintained within barrier tissues at the sites of initial pathogen exposure. We will outline our current understanding of TRM in respect to their generation, functional importance, and how future studies must shed light on how we can exploit TRM to develop the next generation of effective vaccines.

# **GENERATION OF TISSUE-RESIDENT-MEMORY CD8 T CELLS**

CD8 T cells are primed within tissue draining lymph nodes and lymphoid tissues. Once primed, CD8 T cells receive tissuespecific signals that allow them entry into tissues, which at steady-state are normally non-permissive to T cell migration (1). Upon entry into mucosal or skin tissues CD8 T cells take up residence and do not recirculate (2, 3). For T cells to enter the small intestine the integrin α4β7 and chemokine receptor CCR9 are important (4, 5). α4β7 and CCR9 expression is induced by dendritic cell-derived signals like the vitamin A metabolite retinoic acid (6). α4β7 is transiently expressed and the timing of its expression correlates with the window of opportunity for T cell migration into the small intestine (2, 4, 5). Primed T cells that enter mucosal tissues differentiate into TRM in response to tissue-derived signals, not limited to but including TGF-β and IL-15 (5, 7). Tissue-derived signals like TGF-β and IL-15 are not uniquely confined to barrier tissues, as their availability is also important for circulating CD8 T cells. TRM originate from KLRG1− effector cells, are not terminally differentiated, however, express lower levels of CD127 and CD122 than circulating memory CD8 T cells (7, 8). While TRM share a common naïve precursor with blood and SLO memory cells they are inherently different from their circulating counterparts (9). Unlike circulating memory CD8 T cells, TRM maintain expression of CD69 and elevated levels of Granzyme B, attributes akin to effector cells (2, 10, 11). Generation of TRM is dependent on CD69 as overexpression *S1pr1* or deletion of CD69

in CD8 T cells limits TRM formation (7, 12). TRM also upregulate the integrin subunit CD103 whereas circulating memory CD8 T cells remain CD103− (2, 8). A qualitative feature that distinguishes circulating TCM and TEM from TRM is that regardless of infection or anatomical location TRM share a signature of core gene transcripts (7). Within this signature are genes involved in chemotaxis, adhesion, and co-stimulation. Expression of some genes associated with circulating memory cells are decreased, e.g., *Eomes, S1pr1,* and *Ly6C*. The signaling events that regulate the transcriptional programing of TRM is unknown.

# **mTOR AND TRM FORMATION**

The mammalian target of rapamycin (mTOR) kinase is a central regulator of many cell processes, including survival, differentiation, and proliferation. It orchestrates cellular responses to external and internal environmental cues. Signaling through the mTOR pathway is an important event for memory formation (13). During each stage of T cell priming, mTOR is activated i.e., TCR/CD28/IL-12. However, inhibiting mTOR in effector CD8 T cells increases the number of memory CD8 T cells in the circulation by increasing the number of effector CD8 T cells that are CD127hiKLRG1−. This suggests that while mTOR is needed early during activation for effector formation, at some point during the effector phase mTOR could be attenuated or turned off in order for effector cells to progress toward memory differentiation.

We recently reported that accumulation of CD8 T cells in the small intestine and Sowell and Marzo Tissue resident memory and mTOR

FRT is critically dependent on prolonged mTOR signaling (14). We showed that blocking mTOR signaling during the effector phase inhibits the accumulation of effector CD8 T cells in the mucosa thereby limiting TRM formation. This suggests that mTOR controls signals that either direct the migration of CD8 T cells into these barrier tissues or influences their survival. More importantly, because one naive CD8 T cell can give rise to memory in both the SLO and mucosal, our observations indicate that mTOR may regulate the divergence between circulating and residentmemory CD8 T cells. It is also possible that mTOR controls the context in which memory precursors receive signals from cytokines and growth factors. Circulating memory CD8 T cells interact with different stromal and hematopoietic cells than tissue-resident-memory CD8 T cells and this could influence their overall function and survival. For example, IL-15 is an important homeostatic cytokine for memory CD8 T cells, received through a contact-dependent mechanism known as trans-presentation, and for TRM formation (5, 15). Within the memory CD8 T cell populations both IL-15-dependent and IL-15 independent populations have been described. mTOR can be activated by IL-15 and inhibition of mTOR leads to a predominantly IL-15 independent memory population (16, 17).

Many cell types within different tissues produce IL-15 and as a result TRM may receive IL-15 signals more frequently than circulating TEM that may only receive transient IL-15 signals when they circulate through the tissues. While TGF-β induces apoptosis in circulating effector CD8 T cells, TRM generated from effector CD8 T cells rely on TGF-β signals (7, 18). Since mTOR has broad affects on cell physiology and is activated in effector CD8 T cells, mTOR may be central in regulating the unique transcriptional program in TRM. mTOR signaling is mediated by two distinct complexes, mTORC1 and mTORC2 that control responses to environmental cytokine milieus (13). In the absence of mTOR, T cells are unable to respond to cytokines that direct their differentiation (19). The differentiation of naïve CD4 T cells into Th1 and Th2 subsets, requires mTORC1 and mTORC2 signaling, respectively. Loss of mTORC1 signaling in CD4

T cells blocks their ability to upregulate T-bet in the presence of IL-12 (20). IL-12 mediated mTORC1 signaling in CD8 T cells sustains the expression of T-bet and promotes differentiation into effector cells (21). Moreover, inhibiting mTORC1 increases the expression of Eomes and skews memory CD8 T cells toward IL-15 dependence (17). CD4 T cells that lack mTORC2 signaling have diminished responses to IL-4 and IL-13. Less is known about the function of mTORC2 in CD8 T cells. However, given CD4 and CD8 T cells' shared dependence on the same cytokines or cytokine signaling pathways for their differentiation, the role of mTORC2 in CD8 T cell function will be an important direction for future study.

# **ARE TRM REQUIRED FOR PROTECTION?**

A major question is whether TRM positioned at sites of exposure are critical to protect against local re-exposure. The presence of TRM can provide local protection (3). However, circulating memory CD8 T cells can migrate into barrier tissues upon re-challenge and provide protection. However, antigen must first make its way to mucosal draining lymph nodes in order to prime circulating memory CD8 T cells and recruit them to infected tissues. Due to their proximity to infected tissue TRM may be able to respond a pathogen exposure more rapidly than circulating memory CD8 T cells (22). The mucosa is the major route of entry for HIV infection. Within a few hours to days after exposure, HIV can breach the mucosal barrier, infect resting CD4 T cells, and presumably establish latent virus reservoirs (23). In non-human primate models of infection, the latent reservoir is seeded rapidly sometime during the first 3 days of infection creating a big challenge for the immune system (24). The time required for circulating memory CD8 T cells to mount a response may not be quick enough to prevent the establishment of HIV infection. It is now evident that eliminating infected founder cell populations in the area of initial HIV entry is a critical requirement for the immune system to provide protection. Robust TRM responses at the portal of HIV entry may be critical component. Whether TRM can directly kill local infected cells is not explicitly known. The surface area of the mucosa barrier is immense and it

is estimated that for every TRM cell there could be tasked to survey as many as ~103 cells (25). Therefore, it is important to know the concentration of TRM that would constitute a critical mass to provide protection. However, recent work has shown that TRM may do more than kill infected cells. TRM can rapidly respond to local challenge and recruit new antigenspecific memory CD8 T cells from the circulation as well as bystander cell populations (26). Moreover, TRM can activate neighboring NK cells and B cells by providing a local source of IL-2, IFN-γ, and TNF-α (27). Collectively, these recent findings suggest that TRM could provide protection not only by killing of infected cells in barrier tissues, but also by taking part in pathogen sensing and initiation of the immune response. Thus it is important to know whether vaccine prime-boost regimens, which can generate large numbers of TRM, will ultimately lead to protection against mucosa-acquired infections such as HIV and HSV.

## **MANIPULATING TRM FOR CLINICAL BENEFIT**

The observation that upon reactivation TRM are capable of inducing activation of innate cells and even protecting against an antigenically unrelated pathogen may have broader implications for vaccine design (27). It is understood that local priming of CD8 T cells or priming within mucosa draining lymph nodes can generate effector CD8 T cells that can become TRM. Building vaccines that favor priming at these tissue sites and the establishment of longlived TRM, while challenging, is important, and may include the following strategies. Orally administered vaccines can induce CD8 T cells that accumulate within tissues such as the female reproductive tract and small intestine of the gastrointestinal tract, while attractive the longevity and functional attributes of the memory cells generated from this approach need to be further elucidated (28). Prime-boost strategies using local challenge potentially can increase the number of TRM to a level that can provide protection. Identifying key regulators of TRM formation, as candidate adjuvants to more traditional vaccine strategies, in order to re-direct primed effectors to mucosal tissues to generate increased numbers of TRM. However, the

ability of these re-positioned TRM to persist like their counterparts that are generated *in situ* is still an area of research that warrants further investigation. On the other hand, CD8 TRM cells may also play a central role in tissue destruction in organspecific autoimmune disorders. The same factors that are important for the generation of TRM may be potential targets for blocking pathogenic CD8 T cell responses in the small intestine of patients with celiac disease. Our findings that low-doses of rapamycin, a pharmacologic mTOR inhibitor, blocked the accumulation of CD8 T cells within the intestinal mucosa suggest the mTOR pathway as a candidate for therapeutic intervention (14). Moreover, we used a model of T cell mediated enterocyte destruction that may have some similarities to celiac disease pathogenesis. Using this model, we demonstrated that rapamycin was capable of blocking the accumulation of CD8 T cells specific for antigen expressed exclusively within the small intestine. In this model, the autoimmunity becomes fatal upon addition of systemic inflammation and the administration of low-dose rapamycin reversed intestinal destruction and enhanced survival. For tumor immunity one hurdle is positioning enough functional CD8 T cells within the tumor milieu. Interestingly, a small population of memory CD8 T cells has been identified in the spleen and lymph nodes that share characteristics of TRM in that they do not re-circulate, and express CD69 (29). It is of significant interest to determine if an equivalent population of TRM populates the tumor and if so how to manipulate these cells to the point where they have a negative impact on tumor growth.

# **ACKNOWLEDGMENTS**

This research was supported by the National Institutes Health Grants 1R21AI095673-01A1 (Amanda L. Marzo), the Rush Brian Piccolo Cancer Research Fund # 62241 (Amanda L. Marzo), and the Chicago Developmental Center for AIDS Research (D-CFAR), an NIH funded program (P30 AI 082151), which is supported by the following NIH Institutes and Centers (NIAID, NCI, NIMH, NIDA, NICHD, NHLBI, and NCCAM) (Amanda L. Marzo).

# **REFERENCES**

cells that localize to mucosal tissues and form resident memory CD8 T cells is controlled by mTOR. *J Immunol* (2014) **193**(5):2067–71. doi:10.4049/ jimmunol.1400074

innate and adaptive immune responses. *Science* (2014) **346**(6205):98–101. doi:10.1126/science. 1254536

**Conflict of Interest Statement:** The Associate Editor, Kimberly Sue Schluns, declares that despite having collaborated with author Ryan T. Sowell in the past 2 years, there has been no conflict of interest during the handling of this manuscript. 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.

*Received: 09 December 2014; accepted: 20 January 2015; published online: 05 February 2015.*

*Citation: Sowell RT and Marzo AL (2015) Residentmemory CD8 T cells and mTOR: generation, protection,* *and clinical importance. Front. Immunol. 6:38. doi: 10.3389/fimmu.2015.00038*

*This article was submitted to Immunological Memory, a section of the journal Frontiers in Immunology.*

*Copyright © 2015 Sowell and Marzo. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

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