It is well known that the SGs can be a target of a variety of bacterial as well as viral infections, such as mumps and cytomegalovirus (CMV). In the case of CMV, the virus is able to establish latent infection in the SGs and is able to evade CD8+ T cell immunity by downregulating MHC class I molecules (174). Virus-specific CD4+ T cells can control viral production, but are not able to eliminate latently infected cells (175, 176) such that persistent virus is selectively sequestered in the vacuoles of glandular acinar epithelial cells (177, 178). In latently infected individuals, resident populations of antigen-specific CD8+ and CD4+ TRM cells are established in the SGs (179, 180) (**Figure 5**). However, their phenotypes, localization, and the local cues regulating their differentiation into TRM, differ significantly (181). CD4+ TRM cells are located predominantly in the stroma of the SGs and their establishment depends on local antigen (179), presumably due to the upregulation of CD69 that antagonizes S1P1-medaited tissue egress (181). By contrast, CD8+ TRM cells express CD103, and localize predominantly within the epithelium of the acini and ducts (179, 180) (**Figure 5**). Local TGF-β signaling in the SGs is required for upregulation of CD103 on

Figure 5 | TRM niches in the in the salivary gland (SG). CD8+ TRM cells are localized predominantly within the epithelium of the acini and duct. Local TGF-β signaling is required for the formation of CD8+ TRM cells in the SG. By contrast, CD4+ TRM cells are localized predominantly within the stroma of the SG. In contrast to CD8+ TRM cells, cognate antigen plays a key role in the formation of CD4+ TRM cells. TEM cells are passing through the normal interstitium. Red lines indicate the representative niche factors that influence the maintenance of TRM cells. Orange and blue cells indicate CD8+ and CD4+ TRM cells, respectively, unless otherwise stated. A blue line indicates the migratory route. A dashed line indicates potential migratory route. Abbreviations: Ly, lymph vessel; Bl, blood vessel; Ag, antigen; TRM, tissue-resident memory T cells; TEM, effector memory T cells.

CD8+ TRM cells and their localization into the epithelium (179, 180). Because CMV downregulates MHC class I molecules, particularly in infected acinar glandular epithelial cells in the SGs, local antigen does not appear to be required for the formation of CD8+ TRM cells in the SGs (179). Indeed, virus-specific CD8+ T cells can be established in the SGs even in the absence of virus infection in this tissue (6, 182, 183). Furthermore, ongoing presentation of late antigens by non-hematopoietic cells in the LN or by virus-uninfected APC (*via* cross-presentation) during CMV infection results in substantial and sustained expansion of antigen-specific CD8+ T cells in the circulation, a process known as memory inflation (184–187). Some of these memory CD8+ T cells are also converted into TRM cells in the SGs on a continual basis (180). Blockade of CXCR3, or the genetic deletion of either integrin α4β1 or E-cadherin on CD8+ T cells reduces the accumulation of CD8+ TRM cells in the SGs (182, 183, 188), suggesting that these molecules promote the migration of circulating CD8+ T cells to the glandular epithelium. In contrast to the inability of the primary CD8+ T cell response to control the virus infection, CD8+ TRM cells resident in the SGs can confer protection upon recall by eliminating CMV infected non-epithelial cells, where CMV fails to achieve complete downregulation of MHC class I molecule (179).

# Non-Barrier Tissues

#### Brain

Owing to the presence of the blood–brain barrier (BBB), the blood–cerebrospinal fluid (CSF) barrier (BCSFB), and the CSF–brain barrier, the central nervous system (CNS) is regarded as an immune privileged site with severely limited ingress of blood-borne T lymphocytes. Relatively few, if any, T cells are present in the healthy brain parenchyma under noninflammatory conditions (189). Consequently, the aberrant accumulation of T cells in the brain parenchyma has generally been considered to be a pathogenic condition. However, it is now becoming clear that the few peripheral T cells present in the brain in the absence of inflammation play key a role in surveying the CNS and keeping the infectious pathogens in check (190), as the lack of these cells can result in opportunistic infections in the CNS (191).

The choroid plexus (CP) is recognized as a major gateway for peripheral T cell access to the CNS (192, 193). The CP is comprised of fenestrated blood capillaries lacking endothelial tight junctions (192). Thus, the barrier properties of the BCSFB at this site rely only on the monolayer of epithelial cells interconnected by tight junctions—a structure permissive for immune cell transit (192). Consequently, around 150,000–750,000 immune cells are present in the CSF of healthy individuals, and more than 90% of the T cells present are antigen-experienced (193). Recent studies have identified a lymphatic vessel network lining the dural sinuses that drain CSF and allow the transit of immune cells from the adjacent subarachnoid space and brain interstitial fluid to the cervical LN (194, 195). This implies that there is the continual trafficking of TEM cells between CNS (e.g., meninges and FSC) and the circulation (196). Nevertheless, the brain parenchyma essentially lacks lymphatic vessels and is mostly devoid of T cells under steady-state conditions.

Upon infection with neurotropic pathogens, antigen-specific T cells infiltrate the subarachnoid spaces of the meninges as well as the perivascular spaces of the parenchymal post-capillary venule, where specialized APC reside (197, 198) (**Figure 6**). T cells are then activated to proliferate and produce cytokines and chemokines in the infected meninges (199–201). This results in local inflammation, which subsequently disrupts vascular tight junctions and the glia limitans, allowing infiltration of T cells into the parenchyma (190, 198) (**Figure 6**). During this process, the balance of local chemokine production regulates the transmigration of circulating T cells into the brain parenchyma (202). In brief, CXCL12 is constitutively expressed on the basolateral surface of endothelial cell layer in the CNS and is also upregulated during inflammation, which promotes CXCR4+ T-cell recruitment to, and retention within, the perivascular space (203, 204). It is only after the local concentration of CXCL12 declines that effector T cells are able to migrate into the brain parenchyma in response to inflammatory chemokines, such as ligands for CXCR3 (205, 206) and CCR5 (207, 208). In the case of neuroinflammation associated with experimental autoimmune encephalomyelitis, the CXCL10–CXCR3 axis also functions to retain T cells within the perivascular space presumably due to differential inflammatory nature in the perivascular space (209).

After the clearance of a viral infection in the CNS, some of the antigen-specific CD8+ T cells that had been recruited to the brain parenchyma differentiate into TRM cells and become resident in the site (133). The numbers of CD8+ TRM cells that establish residency depends on the pathogen and is presumably linked to the tropism and pathogenesis of each virus (210). For example, following intranasal infection with vesicular stomatitis virus, which infects nerve endings, CD8+ TRM cells form clusters at the site of infection, and are widely distributed throughout the brain parenchyma (133). By contrast, intracerebral infection with LCMV, which infects non-neuronal cells in the brain (i.e., glial cells), CD8+ TRM cell populations are primarily established at brain surface structures, such as meninges and CP (around the ventricles or at anatomical borders between different brain regions) (211). In both cases, these CD8+ TRM cell populations are not pathogenic, but confer protection against reinfection even in the absence of circulating memory CD8+ T cells (211).

Regardless of their location and the nature of the infecting pathogens, brain CD8+ TRM cells can be divided into at least two populations based on their expression of CD103 (133, 211–214). It has been proposed that the initial upregulation of CD103 is largely dependent on the local reactivation of CD8+ T cells with cognate antigen in the brain (it remains elevated following antigen clearance) (133). However, it is clear that Treg-derived TGF-β (215, 216), inflammation, and other undefined local factors (213), can also upregulate CD103 on CD8+ TRM cells in the brain in an antigen-independent manner. These different types of instructive signals may account for the distinct gene expression profiles between CD103+ and CD103− CD8+ TRM cells (212, 214) and the superior effector functions for the former (213, 214). It is noteworthy that retroviral knockdown of CD103 impairs the accumulation of CD8+ TRM cells in the brain, indicating the

to the perivascular spaces. Subsequent migration of cells in the brain parenchyma is promoted by chemokines such as ligands for CXCR3 and CCR5 secreted by astrocytes. CD8+ TRM cells are established in the parenchyma and perivascular spaces of the brain. Parenchymal CD8+ TRM cells are divided by the expression of CD103. Although upregulation of CD103 depends largely on cognate antigen, TGF-β may be involved in this process. PD-1 is highly expressed on both CD103+ and CD103− CD8+ TRM cells in the brain parenchyma independent of either antigen or inflammatory signals. Signaling through PD-1 promotes the establishment of CD8+ TRM cells in the brain parenchyma. IL-7 and IL-15 are available in the perivascular spaces, and sustain the homeostatic proliferation of CD8+ TRM cells in this compartment. TEM cells are passing through the cerebrospinal fluid (CSF). Orange and blue cells indicate CD8+ and CD4+ TRM cells, respectively, unless otherwise stated. Red lines indicate the representative niche factors that influence the maintenance of TRM cells. Blue lines indicate the migratory route. Dashed lines indicate potential impact of niche factors. Abbreviations: Ag, antigen; neg, negative; TRM, tissue-resident memory T cells; TEM, effector memory T cells.

importance of CD103 for the recruitment and/or retention of CD8+ T cells early after infection, probably during transmigration through the BBB. Once recruited to the brain parenchyma, however, CD103 expression has no impact on the localization of CD8+ TRM cells (211), which may be attributed to the lack of E-cadherin expression in the adult brain (217). Taken together, it is possible that CD103 expressed on brain CD8+ TRM cells may reflect the prior acquisition of local education but is not functional as an adhesion molecule.

Programmed cell death protein 1 (PD-1) and CD69 are both expressed on CD8+ TRM cells in the brain (including both CD103+ and CD103− TRM) (213). Although the expression of both molecules on CD8+ TRM in non-CNS sites is generally dependent on repetitive antigen engagement (218), it has been demonstrated that both antigen and inflammation are dispensable for the sustained expression of PD-1 as well as CD69, and programmed cell death ligand 1 (PD-L1) in the brain (213). Furthermore, these cells remain functionally competent under these conditions (213). Interestingly, PD-1 expression on brain CD8+ TRM cells is found to be programmed, as environmental factors in the brain induce extensive demethylation of the *Pdcd1* promoter (which controls PD-1 expression) (213). In addition, genetic deletion of either PD-1 or PD-L1 diminishes the establishment of brain CD8+ TRM cells (219, 220). These findings suggest that signaling through PD-1 is a part of the TRM differentiation program and may be attributed to the PD-1 signaling-induced upregulation of CPT1a, an enzyme necessary for fatty acid β-oxidation that promotes memory differentiation (221, 222). Since upregulation of PD-L1 expression is evident on parenchymal cells (e.g., microglia, astrocytes, and oligodendrocytes) following different types of viral infections in the CNS (223–227), it is reasonable to speculate that PD-1 expression by brain CD8+ TRM cells maintains a tolerable balance between immunopathology and immune control of the virus in the CNS (190).

Reports of Ki-67 expression on brain CD8+ TRM cells following resolution of virus infection suggests that these cells are maintained by homeostatic proliferation (211).  Furthermore, CD8+ TRM cells in the brain parenchyma are less responsive to homeostatic cytokines (212). Interestingly, CD8+ TRM cells in the brain parenchyma, especially the CD103+ population, are not able to survive outside their tissue niche. The irreversible nature of tissue adaptation by CD8+ TRM cells in the brain parenchyma is very different to the situation in the lung airway where CD8+

TRM cells retain the plasticity to adapt to different environmental niches for their survival (133, 228).

#### Liver

The liver is a frontline immune tissue in which antigen-rich blood from the gastrointestinal tract enters *via* the portal vein and is passed through a network of sinusoids (the capillary bed of the liver).  Fenestrated sinusoidal endothelium also enables the direct surveillance of hepatocytes by circulating T cells (231).

Recent studies have demonstrated that liver-resident memory CD8+ T cells are established in the sinusoid following systemic infection or vaccination (232) (**Figure 7**). Liver CD8+ TRM cells in mice are mostly CD69+, CXCR3+, and CXCR6+, but lack the expression of CD103, presumably reflecting the lack of tight junctions in the sinusoidal endothelium. The situation in humans is slightly different since a subset of CD8+ TRM in the human liver are CD103+ in both healthy and hepatitis B virusinfected individuals. In this case, the sequential exposure of the cells to IL-15 and TGF-β induces the development of liveradapted CD103+ CD8+ TRM cells (233). Interestingly, mouse liver CD8+ TRM cells exhibit an amoeboid shape and migrate with a crawling action along the sinusoids, whereas circulating CD8+ TEM cells exhibit a round shape and flow rapidly in the sinusoid (232). Lymphocyte function-associated antigen 1 (LFA-1) has been found to be crucial for the patrolling behavior of liver CD8+ TRM cells in the sinusoid (234). It is also known that Kupffer cells, macrophages, and LSEC in the sinusoid constitutively express CXCL16, a CXCR6 ligand (235–237), which attracts NK cells, another resident cell population in the sinusoid (238). This suggests that liver-resident CD8+ T cells and NK cells share this chemokine niche (239), although competition between these populations for this niche has not been reported. Local antigen presentation is clearly important for the prolonged retention and establishment of CD8+ TRM cells in the sinusoid, as targeting antigen presentation to the hepatocytes in the presence of antigen-specific CD8+ T cells in the circulation leads to the massive accumulation of CD8+ TRM cells in the sinusoid, a strategy termed as "prime and trap" (232). Since local antigen presentation in the liver can trigger the formation of tertiary immune structures known as intrahepatic myeloid cell aggregates for T cell population expansion (iMATE) (240), it is tempting to speculate that such follicle-like structures provide special T cell niches in the liver, especially for CD4+ TRM cells.

#### Kidneys

The kidney is a highly vascularized tissue that is crucial for filtering the blood and removing toxins from the body. Lymphocytes are relatively rare in healthy kidneys, although small numbers of resident immune cells such as DC, macrophages, and T cells can be found in the interstitium under steady-state condition (241, 242). CD8+ TRM cells can persist in extravascular renal compartments following direct (243) or regional infections with pathogens (6, 83, 99, 244), although their precise distribution is not clear (244). While the majority of renal CD8+ TRM cells express CD69, even in the absence of antigen, only small fraction of cells express CD103 (83, 243, 244). The tissue-derived factors that influence the formation of renal CD8+ TRM cells are poorly defined. However, it has been reported that a lack of TGF-β signaling leads to reduction in the formation of CD8+ TRM cells in the kidney (244). This has been attributed to the role of TGF-β signaling in promoting trans-endothelial migration of effector CD8+ T cells by upregulating ligands for E- and P-selectin, including an activated form of CD43, and CXCR3 (244). IL-15 is also known to be essential for the upregulation of CD43 (245), which may explain the defective establishment of renal CD8+ TRM cells in the absence of IL-15 (99).

Figure 7 | TRM niches in the in the liver. CD8+ TRM cells are localized within the sinusoid of the liver. These cells exhibit an amoeboid shape and crawling along the sinusoid dependent on the expression of lymphocyte function-associated antigen 1 (LFA-1). Antigen expressed on the hepatocytes play a key role in the establishment of CD8+ TRM cells in the liver sinusoid. Kupffer cells and NK cells are also resident populations in the sinusoid. CXCL16 secreted by Kupffer cells and liver sinusoidal endothelial cells (LSEC) may promote the retention of TRM cells and NK cells within the sinusoid. TEM cells are passing through the sinusoid without crawling. Orange and blue cells indicate CD8+ and CD4+ TRM cells, respectively, unless otherwise stated. A red line indicates the representative niche factor that influences the maintenance of TRM cells. Blue lines indicate the migratory route. Dashed lines indicate potential impact of niche factors. Abbreviations: Ag, antigen; TRM, tissue-resident memory T cells; TEM, effector memory T cells.

#### White Adipose Tissue (WAT)

While TRM generally function locally to guard the vulnerable sites from reinfection, an interesting exception is the establishment of antigen-specific CD8+ TRM cells in the WAT (246). These cells exhibit a high turnover rate and active metabolism and can augment recall responses generated by non-lipid compartments, suggesting that the WAT functions as a reservoir of TRM cells by improving their functional capacities and longevity. Notably, WAT TRM cells also remodel the physiological function of the WAT, as reactivation of adipose TRM cells lead to a sharp decrease in lipid synthesis. This elevates the antimicrobial responses within the adipose tissues, resulting in synergic immunological crosstalk between the tissue and the TRM cells. Thus, it is of interesting to speculate that, beyond the role as the local sentinel, long-term maintenance of TRM cells may influence the homeostasis and function of each tissue, leading to both beneficial and detrimental consequences.

#### Tumor

It has been reported that CD8+ T cells with a TRM phenotypes (CD103+ and CD49a+) are present in solid tumors (247, 248). Large-scale transcriptome analysis has revealed that CD8+ tumor infiltrating lymphocytes (TIL) exhibit characteristics of TRM cells and it has been observed that CD103+ CD8+ TRM cells from neighboring peripheral tissues can infiltrate into solid tumors (249, 250). Runx3 expression appears to promote the infiltration of CD8+ TRM cells into tumors as *Runx3*-deficient CD8+ T cells failed to accumulate in tumors (90). As with other tissues, local microenvironmental cues promote the acquisition of TRM phenotypes of CD8+ that infiltrate tumor tissues (251). It is important to note, however, that CD8+ TIL with TRM characteristics (termed as CD8+ TRM TIL hereafter) are no longer true "resting" TRM cells as they are located in an effector site where cognate antigen is abundant and typically express checkpoint molecules to regulate their activity (249). This checkpoint molecule expression may be transient, or below suppressive levels, since CD8+ TRM TIL in tumors exhibit superior anti-tumor activities and a positive prognosis has been correlated with the quality and quantity of these cells (248–250, 252–256). It has also been found that CD103+ CD8+ TRM TIL with the strongest CTL activity are located in the border area of the tumor. This contrasts with CD103 negative CD8+ TRM TIL that infiltrate the stroma of the tumor (a potentially highly immune suppressive environment), and mediate weak CTL activity (257). CD103-mediated efficient interaction of CD8+ TRM TIL with tumor cells of epithelial origin also promotes prolonged survival and enhanced CTL activity (251, 254, 258, 259). Based on these findings, the generation of CD8+ TRM cells in neighboring tissues to the tumor is a promising strategy to confer protection against tumor growth (250, 260–263). However, this protection is limited to primary tumors, and not metastases, since CD8+ TRM cells are segregated from the circulation (250).

#### LYMPHOID ORGANS

### Secondary Lymphoid Organs

#### LNs, Spleen

The SLOs have generally been considered a transit site for TCM and TEM cells. In the case of the LN, these cells are transiting from the high endothelial venules and afferent lymphatics, respectively, into the circulation. However, recent studies have demonstrated that there are also small numbers of memory CD4+ and CD8+ T cells that are resident in the LN, spleen, PP, and tonsils without recirculation (264–268). The long-term residency of TRM cells within the SLO has been demonstrated by parabiosis or photoconversion-based cell labeling studies (264, 265, 267, 268). Unlike circulating memory T cells, TRM cells in the SLO share phenotypic characteristics and gene expression profiles with those in the NLT (110), including stable downregulation of S1P1, a key molecule for regulating T-cell egress from the LN (55). Indeed, most TRM cells in the SLO express CD69, which promotes the downregulation of S1P1 (110, 264, 266, 268). Since surface expression of CD69 is generally transient, however, it is likely that repetitive antigen stimulation is required for the maintenance of CD69 expression and the retention of TRM cells in the SLO (110). In this regard, there is considerable evidence that residual antigen persists in the draining LN for several months after vaccination or the resolution of an acute infection and presumably facilitates the accumulation of memory T cells (154–156, 269–272). In addition, a recent study by Beura et al. have demonstrated that some CD8+ TRM cells in the LN are derived from cells that exit the NLT (273), thereby enhancing the accumulation of antigen-specific CD8+ TRM cells in the draining LN.

The distribution of TRM cells in the SLO depends on an antigen niche, as TRM cells are preferentially localized at the common antigen entry sites: the marginal zone and red pulp of the spleen and the subcapsular sinuses of the LN (264) (**Figure 8**). Although the maintenance of murine TRM cells in the SLO is relatively independent of IL-15, signaling *via* IL-15 and TGF-β are known to transcriptionally downregulate S1P1 in human T cells. Indeed, TRM cells in the tonsils are localized specifically near the epithelial barrier where IL-15 is constitutively expressed (266). This is indicative of cytokine niche-dependent compartmentalization of TRM cells within the SLO. Since TCM cells in the SLO are central to pathogen clearance by generating massively increased numbers of secondary effector T cells during a recall response, it will be important to determine the functional contribution of TRM cells in the SLOs during the recall responses. It is possible that TRM cells in the SLO do not actively contribute to the recall response to avoid unnecessary competition with TCM cells, but are strategically positioned to protect the SLO from direct infection with pathogens.

### Primary Lymphoid Organs

#### Thymus

Antigen-specific CD8+ TRM cells have also been found to persist in the thymus, a primary lymphoid organ (274). Thymic CD8+ TRM cells are established following infection with either thymus-tropic or non-tropic pathogens, with considerably higher numbers in the former. As with TRM cells in the peripheral tissues, thymic CD8+ TRM cells exhibit a canonical TRM phenotype (CD69+ CD103+). These cells localize predominantly in the medulla although a few cells lodge in the cortex (**Figure 9**). At least three mechanisms potentially explain the medullary localization of thymic CD8+ TRM cells. First, active TGF-β, which support the

such as the subcapsular sinus in the lymph nodes (A), and the marginal zone and red pulp in the spleen (B). Retention of cells in these compartment is largely dependent on the expression of CD69 in response to antigen, although retention induced by CD69-independent mechanisms is also suspected. Orange cells indicate CD8+ TRM cells unless otherwise stated. Red lines indicate the representative niche factors that influence the maintenance of TRM cells. Abbreviations: Ag, antigen; TRM, tissue-resident memory T cells.

Figure 9 | TRM niches in the in the thymus. CD8+ TRM cells are localized predominantly within the thymic medulla, although some cells are also found in the cortex. A majority of CD8+ TRM cells in the thymus express CD103 and CD69. TGF-β is rich in the medulla, and presumably influences the CD8+ T cell expression of CD103. Since persistent presentation of foreign antigen in the thymus is uncommon, self-antigen may drive the expression of CD69 on thymic CD8+ TRM cells. Orange cells indicate CD8+ TRM cells unless otherwise stated. Dashed lines indicate potential impact of niche factors. Abbreviations: Ag, antigen; cTEC, cortical thymic epithelial cell; mTEC, medullary thymic epithelial cell; TRM, tissue-resident memory T cells.

generation of thymic Treg cells and potentially upregulates T cell expression of CD103, is predominantly localized in the thymic medulla (275). Second, E-cadherin is highly expressed in all thymic epithelial cells (TEC) of both the cortex and medulla (276) and promotes the interaction of TEC with CD103+ thymocytes (277). Third, mature thymocytes express CD69 which induces the downregulation of S1P1 on CD8+ TRM and blocks the departure of the cells *via* the medulla or cortico-medullary junction (278). The factors that induce the upregulation of CD69 on thymic CD8+ TRM cells have not been determined (274). Since the immune activation process strongly inhibits the migration of peripheral DC populations to the thymus to avoid unfavorable induction of acquired tolerance to the invading pathogens (279, 280), it is reasonable to think that thymic CD8+ TRM cells mainly function to protect the thymus, rather than contribute to the recall responses against systemic infections.

#### Bone Marrow (BM)

The BM is another primary lymphoid organ that facilitates the long-term maintenance of memory T cells by providing at least two distinct niches: a quiescence niche, that harbors a majority of quiescent memory T cells, and a self-renewal niche where memory T cells undergo homeostatic proliferation (281). Indeed, large numbers of memory CD8+ and CD4+ T cells accumulate in the BM (282, 283) and most of them express high levels of CD69, a hallmark of TRM cells (164, 284, 285). TGF-β, secreted mainly by megakaryocytes in the BM, regulates the quiescence of memory T cells (286) and CXCL12 produced by reticular stromal cells promotes their co-localization with CXCR4+ memory T cells (287). The reticular stromal cells, as well as myeloid cells, in the BM also provide niche factors for self-renewal such as IL-7 and IL-15 (283, 288, 289).

Recently, Di Rosa and Gebhardt have speculated that memory CD8+ T cells in the BM are a circulating population that is transiting through the BM niches without establishing residence (290). This is largely based on the observation that memory CD8+ T cells derived from the host and partner equilibrate in the BM in parabiosis experiments (65). By contrast, the deposition of memory CD4+ T cells in the BM is relatively stable, as these cells persist in the BM for a long period even after most memory CD4+ T cells disappear from the spleen and LN (283). Interestingly, BM memory CD4+ T cells preferentially home back to the BM after adoptive transfer (283). A fraction of adoptively transferred splenic CD8+ T cells, particularly those with a memory phenotype, also home to the BM (282, 284, 291). These data suggest that circulating memory T cells have high levels of access to BM niches. High levels of access of memory T cells to the BM niches could also explain the low detection of TRM cells in the parabiosis experiments. More analyses are required for precise characterization of TRM cells in the BM.

#### CONCLUDING REMARKS

The regulation, generation, and maintenance of TRM cells depends on two primary cell-extrinsic factors: (i) local signals that enable microenvironmental adaptation of T cells in each tissue and (ii) the availability of tissue-specific anatomical niches. Non-immune cells as well as immune cell populations resident in each microenvironment provide these niche factors. Once established, TRM cells function locally to guard the vulnerable sites from reinfection. Hence, a deep understanding the comprehensive picture of TRM niches is required for the development of tissue-targeted vaccination strategies to effectively generate TRM cells in each tissue. For example, "prime and pull" is a potential vaccination strategy for the skin and FRT, where TRM cells can utilize niches that are originally occupied by other resident cells (37, 127). In sharp contrast, this strategy does not work for the lung due to the absence of preformed niches for TRM cells to displace (134, 158). The creation of *de novo* niches in the lung by "prime and pull plus cognate antigen" partly resolves this problem (5, 134, 158). Antigen-niches also play a role in the establishment of TRM cells in the vascularized tissues of the liver, a strategy referred to as "prime and trap" (232).

### REFERENCES

The description of TRM niches in this review is based primarily on findings from mouse studies with occasional reference to work in humans. It is important to note, however, that the characteristics of TRM cells in these species can vary. For example, the TRM signature in humans is primarily defined by CD69+ expression (292), while CD69 expression is insufficient to infer tissue residence in mice (6, 273). Furthermore, a key transcription factor Hobit that instructs tissue residency is highly expressed by murine TRM cells (62), while its expression is relatively low in human TRM cells (292–294). These, and other, species differences in TRM indicate that many more studies in humans will be necessary for the development of effective vaccines in the clinic.

In summary, the factors regulating the formation of TRM cells in each tissue and each species are far more complex than originally thought, and numerous hurdles exist in generating and maintaining TRM cells in each tissue in terms of the efficacy, safety, and longevity. There is still much to learn.

### AUTHOR CONTRIBUTIONS

ST participated in the concept, wrote the manuscript, and developed the figures.

#### ACKNOWLEDGMENTS

We thank Drs. Makoto Kurachi, Norifumi Iijima, Hiroyuki Taniyama, Katsuro Higiwara, Fumitaka Sato, Takeshi Shimaoka, Kensuke Takada, and Koji Tokoyoda for discussions, and Dr. David L. Woodland for editing the manuscript.

#### FUNDING

This work is supported by Grant-in-Aid for Young Scientists (A) 24689043, and Grant-in-Aid for Scientific Research (C) 16K08850 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and grants from Takeda Science Foundation, Daiichi-Sankyo Foundation of Life Science, Uehara Memorial Foundation, Kanae Foundation for the Promotion of Medical Science, The Waksman Foundation of Japan, Kato Memorial Bioscience Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Life Science Foundation of Japan, Japan Foundation for Pediatric Research, The Naito Foundation, and SENSHIN Medical Research Foundation.

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**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Takamura. 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) and the copyright owner 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.*

# Memory Inflation Drives Tissue-Resident Memory CD8**+** T Cell Maintenance in the Lung After Intranasal Vaccination With Murine Cytomegalovirus

*Kaitlyn M. Morabito1,2, Tracy J. Ruckwardt1 , Erez Bar-Haim1,3, Deepika Nair1 , Syed M. Moin1 , Alec J. Redwood4 , David A. Price5,6 and Barney S. Graham1 \**

#### *Edited by:*

*Fathia Mami-Chouaib, Institut National de la Santé et de la Recherche Médicale (INSERM), France*

#### *Reviewed by:*

*Steven Varga, University of Iowa, United States Georg Gasteiger, Julius-Maximilians-Universität, Germany*

#### *\*Correspondence:*

*Barney S. Graham<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 26 March 2018 Accepted: 27 July 2018 Published: 14 August 2018*

#### *Citation:*

*Morabito KM, Ruckwardt TJ, Bar-Haim E, Nair D, Moin SM, Redwood AJ, Price DA and Graham BS (2018) Memory Inflation Drives Tissue-Resident Memory CD8+ T Cell Maintenance in the Lung After Intranasal Vaccination With Murine Cytomegalovirus. Front. Immunol. 9:1861. doi: 10.3389/fimmu.2018.01861*

*1Viral Pathogenesis Laboratory, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States, 2Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC, United States, 3Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, Israel, 4 Institute for Immunology and Infectious Diseases, Murdoch University, Perth, WA, Australia, 5Division of Infection and Immunity, Cardiff University School of Medicine, Cardiff, United Kingdom, 6Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States*

Tissue-resident memory T (TRM) cells provide first-line defense against invading pathogens encountered at barrier sites. In the lungs, TRM cells protect against respiratory infections, but wane more quickly than TRM cells in other tissues. This lack of a sustained TRM population in the lung parenchyma explains, at least in part, why infections with some pathogens, such as influenza virus and respiratory syncytial virus (RSV), recur throughout life. Intranasal (IN) vaccination with a murine cytomegalovirus (MCMV) vector expressing the M protein of RSV (MCMV-M) has been shown to elicit robust populations of CD8+ TRM cells that accumulate over time and mediate early viral clearance. To extend this finding, we compared the inflationary CD8+ T cell population elicited by MCMV-M vaccination with a conventional CD8+ T cell population elicited by an MCMV vector expressing the M2 protein of RSV (MCMV-M2). Vaccination with MCMV-M2 induced a population of M2-specific CD8+ TRM cells that waned rapidly, akin to the M2-specific CD8+ TRM cell population elicited by infection with RSV. In contrast to the natural immunodominance profile, however, coadministration of MCMV-M and MCMV-M2 did not suppress the M-specific CD8+ T cell response, suggesting that progressive expansion was driven by continuous antigen presentation, irrespective of the competitive or regulatory effects of M2-specific CD8+ T cells. Moreover, effective viral clearance mediated by M-specific CD8+ TRM cells was not affected by the coinduction of M2-specific CD8+ T cells. These data show that memory inflation is required for the maintenance of CD8+ TRM cells in the lungs after IN vaccination with MCMV.

Keywords: CD8+ T cells, cytomegalovirus, memory inflation, respiratory syncytial virus, tissue-resident memory, vaccine

# INTRODUCTION

Tissue-resident memory T (TRM) cells protect against invading pathogens in barrier tissues by direct killing of infected cells and by recruitment of other immune effector cell populations into the tissue. Much work has been done in recent years to characterize the migration pattern, function, and phenotype of TRM cells in various anatomical locations (1–4). It has become clear that TRM cells are heterogeneous, and that the requirements for localization and maintenance differ across tissues (4–9). In the lungs, TRM cells have been shown to mediate immune protection against respiratory syncytial virus (RSV) (10–12) and heterosubtypic cross-protection against influenza virus (13–16). TRM cells are also important for immune protection against cancer (17–23). In particular, TRM cells have been shown to enhance the efficacy of intranasally administered cancer vaccines in mouse orthotopic head and neck tumor models (23). The abundance of TRM cells in malignant lung tumors further correlated with survival in humans (23). However, lung-resident TRM cells tend to wane over time, potentially reflecting a harsher and more dynamic environment compared with other barrier tissues (13, 14, 16, 24, 25). This progressive loss of TRM cells likely explains why recurrent infections with RSV and influenza virus occur throughout life. Vaccination strategies aimed at maintaining high levels of TRM cells in the lungs may therefore enhance immunity against respiratory pathogens and cancers.

Cytomegalovirus (CMV) has been shown to elicit robust populations of TRM cells in some tissues (26, 27). The persistent nature of CMV leads to a unique phenomenon among memory CD8+ T cells, which has been well characterized in mouse models using murine cytomegalovirus (MCMV). Specifically, MCMV infection generates two distinct populations of memory CD8+ T cells, termed conventional and inflationary (28–32). Conventional CD8+ T cell populations expand during acute infection and then contract, whereas inflationary CD8+ T cell populations, which may not predominate in the early phase, continue to accumulate over time within the effector memory (EM) compartment. The ability to drive memory inflation may explain why CMV vectors have shown promise as vaccine candidates, protecting against various cancers and infectious agents and providing effective immunocontraception (33–41).

Several factors determine whether a particular epitope will elicit conventional or inflationary CD8+ T cell populations. For inflationary memory responses, the source protein must be transcribed during latency, a feature that depends primarily on location within the genome (42). In addition, the derived epitope may require processing by constitutive proteasomes, because antigen presentation occurs predominantly on the surface of nonhematopoietic cells, which lack immunoproteasomes (43, 44). Interclonal competition may also play a role, given the observation that high-avidity clonotypes are preferentially selected for inflation during MCMV infection (45–47). Similar findings have been reported in the setting of human CMV infection (41, 48–50). Other potential contributors include epitope-dependent requirements for co-stimulation and CD4+ T cell help (51–55). Memory inflation is therefore difficult to predict, even in welldefined mouse models, yet a detailed understanding of this phenomenon is critical for the design of effective vaccines that deliver protective antigens vectored by CMV.

Infection of CB6F1 mice with RSV elicits CD8+ T cell responses that reproducibly target an immunodominant epitope from the M2 protein (Kd /M282–90) and a subdominant epitope from the M protein (Db /M187–195) (56). The M-specific CD8+ T cell population typically incorporates high-avidity clonotypes expressing private T cell receptors with characteristic sequence motifs, leading to greater levels of cytokine production and more effective killing of virus-infected targets in side-by-side comparisons with the M2-specific CD8+ T cell population (57–59). In addition, M-specific CD8+ T cells regulate the magnitude of the otherwise numerically dominant M2-specific CD8+ T cell population, an effect that mitigates the immunopathology associated with acute RSV infection (57).

Intranasal (IN) vaccination with an MCMV vector expressing the M protein of RSV (MCMV-M) has been shown to generate a robust population of M-specific CD8+ TRM cells with an effector/EM phenotype and augment early viral control relative to vaccination with MCMV alone or MCMV-M inoculated *via* the intraperitoneal (IP) route (60). In this study, we characterized the M2-specific CD8+ T cell response to IN vaccination with an MCMV vector expressing the M2 protein of RSV (MCMV-M2). Vaccination with MCMV-M2 induced a population of M2-specific CD8+ TRM cells in the lungs that subsequently waned over time, whereas vaccination with MCMV-M induced a population of M-specific CD8+ TRM cells in the lungs that subsequently inflated over time. Coadministration of both vaccines diminished the M2-specific CD8+ T cell response, but not the M-specific CD8+ T cell response, during the acute phase of infection, but had no impact on the magnitude of the conventional M2-specific CD8+ T cell population or the inflationary M-specific CD8+ T cell population during the chronic phase of infection. Moreover, the inclusion of MCMV-M2 neither enhanced nor impaired the protective effects of vaccination with MCMV-M alone in challenge experiments with RSV.

### MATERIALS AND METHODS

#### Mice

All experiments were conducted with age-matched (6–10 weeks) female CB6F1/J mice (Jackson Laboratories, Bar Harbor, ME, USA). Mice were maintained under specific-pathogen-free conditions on standard rodent chow and water supplied *ad libitum* in the Animal Care Facility at the National Institute of Allergy and Infectious Diseases. This study was carried out in accordance with the recommendations and guidelines of the NIH Guide to the Care and Use of Laboratory Animals. The protocol was approved by the Animal Care and Use Committee of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Mice were housed in a facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Animal procedures were conducted in strict accordance with all relevant federal and National Institutes of Health guidelines and regulations.

## Cell Lines

CB6F1 mouse embryonic fibroblasts (MEFs) were isolated as described previously (60). MEFs were cultured in Advanced Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS), 2 mM glutamine, 10 U/ml penicillin G, 10 µg/ml streptomycin sulfate, and 0.1 M HEPES (DMEM-10). Human epithelial type 2 (HEp-2) cells were cultured in Eagle's Minimal Essential Medium (MEM; Invitrogen) containing 10% FBS, 2 mM glutamine, 10 U/ml penicillin G, 10 µg/ml streptomycin sulfate, and 0.1 M HEPES (MEM-10).

#### Viruses and Infection

Recombinant MCMVs were made using a bacterial artificial chromosome (BAC) system as described previously (35). Briefly, the M and M2 proteins from RSV were inserted into the IE2 gene of the K181Δm157 strain of MCMV using two-step allele replacement. BACs were extracted from *E. coli* using a NucleoBond Xtra Maxi Prep Kit (Clontech, Mountain View, CA, USA). MEFs were transfected with recombinant BACs by calcium phosphate precipitation (Clontech) as described previously (35). Single plaques were isolated by serial dilution after viral passage and selected based on excision of the BAC cassette determined by loss of GFP and confirmed by PCR. Viral stocks were made by sonication of infected MEFs, and plaque assays were performed in triplicate on CB6F1 MEFs. Mice were vaccinated IN with 3 × 105 PFU of recombinant MCMV-M and/or MCMV-M2 in 100 µl of DMEM-10 under isoflurane anesthesia (3%). For RSV challenge, stocks were generated from the A2 strain by sonication of infected HEp-2 monolayers as described previously (61). Mice were challenged IN with 2 × 106 PFU of RSV in 100 µl of MEM-10 under isoflurane anesthesia (3%). All mice were euthanized *via* the administration of pentobarbital (250 mg/kg).

### Intravascular Staining and Flow Cytometry

Mice were injected intravenously (IV) with 3 µg of anti-CD45 (BD Biosciences, San Jose, CA, USA). Five minutes after intravascular staining, mice were euthanized with pentobarbital, and the lungs were harvested at various time points. Lymphocytes were isolated by physical disruption of tissue using a GentleMACs Machine (Miltenyi Biotec, San Diego, CA, USA) and separated using density gradient centrifugation with Fico-LITE (Thermo Fisher Scientific, Waltham, MA, USA). Isolated mononuclear cells were washed with phosphate-buffered saline (PBS) and resuspended in fluorescence-activated cell sorting buffer (PBS supplemented with 1% FBS and 0.05% sodium azide). Cells were stained with directly conjugated antibodies specific for the lineage markers CD3 (145-2C11) and CD8 (53-6.7) (BD Biosciences) and the memory markers CD44 (IM7), CD62L (MEL-14), CD127 (A7R34), KLRG1 (2F1/KLRG1), CD69 (H1.2F3), and CD103 (M290) (BD Biosciences or BioLegend, San Diego, CA, USA). Dead cells were excluded from the analysis using LIVE/ DEAD Fixable Aqua (Invitrogen). Antigen-specific CD8+ T cells were identified using Db /M187–195 (RSV M) or Kd /M282–90 (RSV M2) tetramers (MBL, Woburn, MA, USA). For validation of intravascular staining, cells were labeled with directly conjugated antibodies specific for CD3 (145-2C11), CD11c (N418), CD64 (X54-5/7.1), SiglecF (E50-2440), and CD11b (M1/70) (BD Biosciences or BioLegend). Data were acquired using an LSR II flow cytometer (BD Biosciences) and analyzed with FlowJo software version 9.9.6 (TreeStar, Ashland, OR, USA). Memory phenotypes were further analyzed using Pestle version 1.6.2 and SPICE version 6.0 (http://exon.niaid.nih.gov/spice/).

# ONX-0914 Inhibition Study

Mice were treated subcutaneously on days 0, 2, 4, and 6 with 2, 6, or 10 mg/kg of ONX-0914 (PR-957; Selleck Chemical, Houston, TX, USA) or vehicle control (10% captisol in 10 mM sodium citrate). On day 0, mice were infected IN as described above with 2 × 106 PFU of RSV. On day 7, mice were euthanized with pentobarbital, and the lungs were harvested and processed as described above.

#### Plaque Assay

Lungs were weighed and quick-frozen in 10% MEM-10, and plaque assays were performed as described previously (62). Briefly, thawed lung tissue was dissociated using a GentleMACs Machine (Miltenyi Biotec). Cell suspensions were pelleted to remove cellular debris, and clarified supernatants were serially diluted and inoculated in triplicate on 80% confluent HEp-2 cell monolayers. After rocking for 1 h at room temperature, monolayers were overlaid with 0.75% methyl cellulose in MEM-10 and incubated at 37°C. Cells were fixed with 10% buffered formalin and stained with hematoxylin and eosin on day 4. Plaques were counted and expressed as Log10 PFU/g of lung tissue. The limit of detection was 1.8 Log10 PFU/g.

### Statistical Analysis

Statistical analyses were performed using a one-way or two-way ANOVA as appropriate for multiple comparisons (GraphPad Prism, San Diego, CA, USA). Memory phenotypes were compared using a permutation test (10,000 rounds) in SPICE version 6.0 (http://exon.niaid.nih.gov/spice/).

# RESULTS

# IN Vaccination With MCMV-M2 Elicits More Lung-Resident M2-Specific CD8**+** T Cells Than IP Vaccination

We and others have demonstrated that IN vaccination is necessary to elicit TRM cells in the lungs (19, 23, 60). In particular, our earlier work showed that IN vaccination with MCMV-M elicited more M-specific CD8+ T cells in the lung parenchyma than IP vaccination with MCMV-M (**Figure 1A**) (60). To extend this finding, we vaccinated mice with MCMV-M2 *via* the IN or IP route and used intravascular staining in conjunction with Kd / M282–90 tetramers to analyze M2-specific CD8+ T cell responses in the blood and the lung parenchyma after 1 week. The intravascular staining protocol was validated in the context of IN vaccination to ensure that direct infection of the lungs did not lead to increased permeability due to inflammation (Figure S1 in Supplementary Material). Akin to the differences observed

Figure 1 | Intranasal (IN) vaccination with murine cytomegalovirus (MCMV)-M2 elicits more lung-resident M2-specific CD8+ T cells than intraperitoneal (IP) vaccination. (A–E) Mice were vaccinated with MCMV-M or MCMV-M2 *via* the IN or IP route. Intravascular staining was used in conjunction with Db/M187–195 and Kd/M282–90 tetramers to quantify epitope-specific CD8+ T cells in the lung tissue and blood after 1 week. (A) Gating strategy used to identify M-specific and M2-specific CD8+ T cells in the tissue and blood of the lungs. (B) Frequency and (C) number of M-specific CD8+ T cells in the tissue and blood of lungs 1 week after MCMV-M vaccination. (D) Frequency and (E) number of M2-specific CD8+ T cells in the tissue and blood of the lungs 1 week after MCMV-M2 vaccination. Bars indicate mean ± SEM (*n* = 5 mice/group). \*\*\*\**P* < 0.0001, \*\**P* < 0.01, \**P* < 0.05 by two-way ANOVA. Data are shown from one experiment and representative of two independent experiments.

after vaccination with MCMV-M (**Figures 1B,C**), we found that IN vaccination with MCMV-M2 induced significantly more lung-resident M2-specific CD8+ T cells than IP vaccination with MCMV-M2, both in terms of frequency (*P* < 0.01; **Figure 1D**) and number (*P* < 0.05; **Figure 1E**). By contrast, IP vaccination with MCMV-M2 elicited higher frequencies of M2-specific CD8+ T cells in the blood (*P* < 0.0001, **Figure 1D**) and in total (*P* < 0.05), but similar numbers of M2-specific CD8+ T cells in the blood and in total. We therefore focused on IN vaccination in our efforts to induce and maintain lung-resident CD8+ T cells.

### The M-Specific CD8**+** T Cell Population Inflates, Whereas the M2-Specific CD8**+** T Cell Population Contracts, After Vaccination With MCMV

Next, we used a similar approach to evaluate CD8+ T cell responses at weeks 1, 8, and 16 after vaccination with MCMV-M or MCMV-M2 alone or a combination of MCMV-M and MCMV-M2. Intravascular staining was used as above in conjunction with Db /M187–195 and Kd /M282–90 tetramers to quantify epitope-specific CD8+ T cells in the blood and lung parenchyma. MCMV-M administered either alone or together with MCMV-M2 generated an M-specific CD8+ T cell population that inflated between weeks 1 and 8 (*P* < 0.0001) and remained stable through week 16 (**Figure 2A**). This trend was observed in the lung tissue and blood (*P*< 0.0001; **Figures 2B,C**). By contrast, M2-specific CD8+ T cells in the lung tissue and blood contracted over time (*P* < 0.0001; **Figures 2D–F**), irrespective of coadministration with MCMV-M. After RSV infection, which generates only conventional memory responses as a consequence of self-limited

Figure 2 | The M-specific CD8+ T cell population inflates, whereas the M2-specific CD8+ T cell population contracts, after vaccination with murine cytomegalovirus (MCMV)*.* (A–F) Mice were infected with respiratory syncytial virus (RSV) or vaccinated with MCMV-M or MCMV-M2 alone or a combination of MCMV-M and MCMV-M2 *via* the intranasal route. Intravascular staining was used in conjunction with Db/M187–195 and Kd/M282–90 tetramers to quantify M-specific (A–C) and M2-specific (D–F) CD8+ T cells in the lung tissue and blood at weeks 1 (W1), 8 (W8), and 16 (W16). Total (A,D) denotes all tetramer+ CD8+ T cells regardless of location. Bars indicate mean ± SEM (*n* = 5 mice/group). \*\*\*\**P* < 0.0001, \*\*\**P* < 0.001, \*\**P* < 0.01, \**P* < 0.05 by two-way ANOVA. Data are shown from one experiment and representative of two independent experiments.

antigen production, the M-specific and M2-specific CD8+ T cell populations both contracted dramatically between weeks 1 and 8 in the lung tissue and blood (*P* < 0.001; **Figures 2A–F**). Similar epitope-specific patterns were observed when assessing T cell frequency in the lung and spleen (Figure S2 in Supplementary Material). In addition, the MCMV-encoded M38-specific CD8+ T cell response was largely equivalent among experimental groups, suggesting that the observed loss of M2-specific CD8+ T cells over time was not attributable to clearance of MCMV-M2. Thus, the M-specific CD8+ T cell population is inflationary, whereas the M2-specific CD8+ T cell population is not inflationary, after vaccination with MCMV.

One week after vaccination, coadministration of MCMV-M and MCMV-M2 elicited an epitope-specific hierarchy equivalent to that observed after RSV infection, with a dominant CD8+ T cell response to Kd /M282–90 and a subdominant CD8+ T cell response to Db /M187–195 (**Figures 2A,D**). At weeks 8 and 16, this hierarchy was inverted as a consequence of M-specific CD8+ T cell inflation and M2-specific CD8+ T cell contraction (**Figures 2A,D**). Coadministration of MCMV-M and MCMV-M2 did not alter the number or frequency of M-specific CD8+ T cells in the blood or the tissue at any time point relative to vaccination with MCMV-M alone (**Figures 2B,C**; Figure S2B,C in Supplementary Material). By contrast, coadministration of MCMV-M and MCMV-M2 dampened the frequency, but not the overall magnitude, of the M2-specific CD8+ T cell response at week 1 (*P* < 0.01), but not at weeks 8 and 16 (**Figure 2D**; Figure S2D in Supplementary Material). This effect was anatomically discrepant. Specifically, coadministration of MCMV-M and MCMV-M2 did not significantly reduce the number or frequency of M2-specific CD8+ T cells in the lung tissue (**Figure 2E**; Figure S2E in Supplementary Material), but did significantly reduce the number and frequency of M2-specific CD8+ T cells in the blood at weeks 1 and 8 relative to vaccination with MCMV-M2 alone (*P* < 0.01; **Figure 2F**; Figure S2F in Supplementary Material). No significant differences in the frequency of M2-specific CD8+ T cells were observed after contraction of the response at week 16 (**Figures 2D–F**). The reduction of M2-specific CD8+ T cells at the acute time point after coadministration of MCMV-M and MCMV-M2 was not unexpected, because competition between the M-specific and M2-specific CD8+ T cell populations has been demonstrated after RSV infection of CB6F1 mice (57).

#### The M2 Epitope Is Preferentially Generated by the Immunoproteasome

Memory inflation likely requires epitope generation *via* the constitutive proteasome, because antigen processing and presentation are thought to occur predominantly by non-hematopoietic cells, which lack immunoproteasomes (43, 44). To determine if proteasomal processing impacted the M-specific or M2-specific CD8+ T cell responses, we infected mice with RSV and treated them with the immunoproteasome inhibitor ONX-0914 on days 0, 2, 4, and 6 at doses of 2, 6, or 10 mg/kg. On day 7, we evaluated M-specific and M2-specific CD8+ T cells in the lungs. Treatment with ONX-0914 significantly reduced the frequency and number of M2-specific CD8+ T cells, but not M-specific CD8+ T cells, in a dose-dependent manner (**Figures 3A,B**). These data suggest that the M2 peptide is preferentially generated by the immunoproteasome, whereas the M peptide is preferentially generated by the constitutive proteasome, which is unaffected by ONX-0914.

### IN Vaccination With MCMV Elicits CD8**+** TRM Cells

A previous study demonstrated that IN vaccination with MCMV-M generated a robust population of TRM cells, identified by expression of CD103 (60). However, it has also been shown that not all TRM cells express CD103 (7). We therefore used intravascular staining to quantify M-specific and M2-specific TRM cells in the lung parenchyma based on expression of CD69 and CD103 after infection with RSV or vaccination with MCMV-M or MCMV-M2 alone or a combination of MCMV-M and MCMV-M2. The administration of MCMV-M, either alone or together with MCMV-M2, generated a substantial population of CD69+ TRM cells that was largely maintained between weeks 8 and 16, and significantly outnumbered the corresponding population

Figure 4 | Intranasal (IN) vaccination with murine cytomegalovirus (MCMV) elicits CD8+ tissue-resident memory T (TRM) cells. (A–E) Mice were infected with respiratory syncytial virus (RSV) or vaccinated with MCMV-M or MCMV-M2 alone or a combination of MCMV-M and MCMV-M2 *via* the IN route. Intravascular staining was used in conjunction with Db /M187–195 and Kd /M282–90 tetramers to quantify M-specific (A,B,D,F) and M2-specific (A,C,E,G) CD8+ T cells in the lung tissue at weeks 8 (W8) and 16 (W16). (A) Representative flow cytometry plots showing expression of CD69 and CD103 on epitope-specific CD8+ T cells in the lung parenchyma at week 8. (B,D) The number of M-specific CD103+CD69+ TRM cells (B) and CD103−CD69+ TRM cells (D) elicited by infection with RSV or vaccination with MCMV-M alone or together with MCMV-M2. (C,E) The number of M2-specific CD103+CD69+ TRM cells (C) and CD103−CD69+ TRM cells (E) elicited by infection with RSV or vaccination with MCMV-M2 alone or together with MCMV-M. (F,G) Percentage of CD103+ and CD103− M-specific CD69+ TRM cells (F) and M2-specific CD69+ TRM cells (G). \*\*\*\**P* < 0.0001 by two-way ANOVA. Bars indicate mean ± SEM (*n* = 5 mice/group). Data are shown from one experiment and representative of two independent experiments.

of CD69+ TRM cells induced by RSV infection at both time points (*P* < 0.0001; **Figures 4A,B,D**). By contrast, the M2-specific CD69+ TRM population significantly decreased between weeks 8 and 16, irrespective of M2 protein expression *via* MCMV or RSV (*P*< 0.01; **Figures 4A,C, E**). There was no difference in the number of M2-specific TRM cells elicited by vaccination with MCMV-M2 or infection with RSV. As the M-specific TRM population induced by MCMV was maintained in the lungs and the M2-specific TRM population induced by MCMV waned in the lungs, there were significantly more M-specific TRM cells than M2-specific TRM cells in the lung parenchyma at weeks 8 and 16 (*P* < 0.0001; **Figures 4B–E**). In the context of RSV infection, however, there were significantly more M2-specific TRM cells than M-specific TRM cells in the lung parenchyma at both time points (*P* < 0.05; **Figures 4B–E**). Coadministration of MCMV-M and MCMV-M2 did not affect the number of M-specific or M2-specific TRM cells at either time point compared with the administration of MCMV-M or MCMV-M2 alone (**Figures 4B–E**).

Next, we assessed the expression of CD103 on CD69+ TRM cells. After vaccination with single MCMV vectors, a higher proportion of M-specific CD8+ T cells coexpressed CD69 and CD103 compared with M2-specific cells at week 8 (84.6 vs. 28.1%; *P*< 0.0001) and week 16 (74.2 vs. 30.9%; *P*< 0.05) (**Figures 4F,G**). A similar trend was observed after coadministration of MCMV-M and MCMV-M2. At week 8, the vaccine-induced M-specific TRM population also contained a significantly higher proportion of cells expressing CD103 than the M-specific TRM population elicited by RSV infection (84.6% for MCMV-M and 83.9% for MCMV-M + MCMV-M2 vs. 55% for RSV; *P* < 0.0001).

These data show that inflation of the M-specific CD8+ T cell population elicited by vaccination with MCMV enhances the frequency and number of TRM cells relative to acute infection with RSV. By contrast, M2-specific CD8+ TRM cells were induced at similar levels irrespective of M2 protein expression *via* MCMV or RSV. It is also notable that a larger fraction of M-specific CD69+ TRM cells elicited by vaccination with MCMV coexpressed CD103 compared with either M2-specific CD69+ TRM cells elicited by vaccination with MCMV or TRM cells of either specificity elicited by infection with RSV.

### M-Specific and M2-Specific CD8**+** T Cells Are Phenotypically Distinct in the Lung Tissue and Blood After Vaccination With MCMV

Inflationary and conventional epitope-specific CD8+ T cell populations have previously been shown to differ phenotypically after IP infection with MCMV (63). In this context, inflationary memory cells are predominantly CD127−KLRG1+ effectors, while conventional memory cells display a more CD127+CD62L+ central memory (CM)-like phenotype. This pattern is recapitulated after IP vaccination with MCMV-M. However IN vaccination with MCMV-M induces a CD8+ T cell population with predominantly effector and EM phenotypes (60). We therefore analyzed the phenotype of antigen-specific CD8+ T cells elicited by MCMV-M and/or MCMV-M2 vaccination at 8 weeks post-vaccination. We categorized the RSV-specific CD8+ T cell populations as CM, EM, effector, or KLRG1+ effectors (KLRG1+) (**Figure 5**). Populations were defined as follows: all: tetramer+ CD44+; CM: CD127+KLRG1−CD62L+; EM: CD127+KLRG1−CD62L−; effector: CD127−KLRG1−CD62L−; KLRG1+ effector: CD62L−KLRG1+. Overall, there were no obvious phenotypic differences when the MCMV vectors were administered alone or in combination (**Figure 5B**). By contrast, distinct phenotypes were observed across anatomical compartments for both the M-specific and M2-specific CD8+ T cell populations, with higher frequencies of KLRG1+ effectors (yellow) and CM cells (blue) and lower frequencies of EM cells (green) in the blood compared with the tissue (*P* < 0.05). A comparison of M-specific and M2-specific CD8+ T cells in the blood and tissue also showed that these antigen-specific populations were comprised of different proportions of memory subsets (**Figure 5**, *P* < 0.05). In the blood, the M2-specific CD8+ T cell population incorporated larger fractions of CM (blue) and KLRG1+ effectors (yellow) and smaller fractions of effector (orange) and EM (green) cells than the M-specific CD8+ T cell population. Although statistically significant, the differences between the M-specific and M2-specific CD8+ T cell population were more subtle in the tissue. Interestingly, we observed higher levels of CD44 expression on CD8+ T cells in the lung tissue compared with CD8+ T cells in the blood, irrespective of antigen specificity and vaccination modality (**Figure 5C**). When parsed out by location, expression of CD44 by M-specific and M2-specific CD8+ T cells was relatively high compared with the corresponding bulk CD8+ T cell populations in the blood and tissue of the lungs (Figure S3 in Supplementary Material).

### MCMV-Elicited TRM Cells Expedite Viral Clearance After Infection With RSV

To evaluate the biological relevance of these observations, we challenged mice with 2 × 106 PFU of RSV delivered *via* the IN route 16 weeks after vaccination with MCMV-M, MCMV-M2, or a combination of MCMV-M and MCMV-M2. Viral loads were measured on days 3 and 5 after infection with RSV. On day 3, mice vaccinated with MCMV-M or MCMV-M together with MCMV-M2 exhibited significantly lower viral loads in the lungs compared with mice vaccinated with the MCMV vector alone (*P* < 0.01 and *P* < 0.05, respectively; **Figure 6A**). By contrast, vaccination with MCMV-M2 did not lead to a significant reduction in viral load on day 3. All vaccination regimens significantly reduced viral loads on day 5 relative to the MCMV vector alone (*P* < 0.0001 for MCMV-M, *P* < 0.01 for MCMV-M2, *P* < 0.0001 for MCMV-M + MCMV-M2; **Figure 6B**). However, simultaneous vaccination with MCMV-M and MCMV-M2 did not enhance viral clearance relative to vaccination with MCMV-M alone (**Figure 6B**).

### Inflation of the M-Specific CD8**+** T Cell Population Alters Immunodominance After Challenge With RSV

In further experiments, we assessed the frequency of antigenspecific CD8+ T cells in the lung parenchyma on day 5 after challenge with RSV (**Figures 6C,D**). Mice vaccinated with the MCMV vector alone harbored relatively few M-specific or M2-specific

Figure 5 | Phenotype of M-specific and M2-specific CD8+ T cells elicited by murine cytomegalovirus (MCMV) vaccination. Mice were vaccinated with MCMV-M or MCMV-M2 alone or a combination of MCMV-M and MCMV-M2 *via* the IN route. Intravascular staining was used in conjunction with Db/M187–195 and Kd/M282–90 tetramers to identify M-specific and M2-specific CD8+ T cells in the blood and tissue of the lungs at week 8. (A) Gating strategy for phenotypic analysis. Populations were defined as follows: CD127+KLRG1−CD62L+ [central memory (CM)]; CD127+KLRG1−CD62L− [effector memory (EM)]; CD127−KLRG1−CD62L− (effector); and KLRG1+CD62L− (KLRG1+ effector). (B) The proportions of CM cells (blue), EM cells (green), effectors (orange), and KLRG1+ effectors (yellow) in the lungs are shown for each specificity. (C) CD44 expression on M-specific, M2-specific, and all CD8+ T cells in the tissue and blood of the lungs. \**P* ≤ 0.05, \*\**P* < 0.01 by permutation test (SPICE). Data are shown from one experiment (*n* = 5/group) and representative of two independent experiments.

CD8+ T cells in the lungs, but the M2-specific population was immunodominant, as typically observed in unvaccinated mice after infection with RSV. As expected, mice vaccinated with MCMV-M or MCMV-M2 alone mounted immunodominant CD8+ T cell responses to the corresponding vaccine antigens, whereas mice vaccinated with both MCMV-M and MCMV-M2 displayed very high frequencies of M-specific CD8+ T cells relative to M2-specific CD8+ T cells, inverting the natural immunodominance hierarchy observed after infection with RSV. This finding may explain why the addition of MCMV-M2 did not enhance the protective effects of vaccination with MCMV-M alone in response to challenge with RSV.

# DISCUSSION

Vaccination with MCMV-M *via* the IN route has been shown to generate a robust population of M-specific CD8+ TRM cells in the lungs that subsequently inflates over time (60). To extend this finding, we evaluated MCMV vaccine-induced CD8+ T cell responses to the immunodominant M2 epitope. We found that IN vaccination with MCMV-M2 induced a conventional memory response, but failed to establish a stable population of lung-resident M2-specific CD8+ TRM cells. Moreover, coadministration of MCMV-M and MCMV-M2 inverted the natural immunodominance hierarchy, but did not significantly impact the generation of M-specific or M2-specific CD8+ TRM cells. As a consequence, the protective effects of vaccination with MCMV-M were neither impeded nor enhanced by the addition of MCMV-M2.

Memory inflation is essential for the maintenance of lungresident CD8+ TRM cell populations. In the setting of self-limiting viral infections of the respiratory tract, conventional epitopes induce populations of CD8+ TRM cells in the lung parenchyma that wane over time (14). Our data further show that persistent antigen expression is insufficient to overcome this decline, consistent with the findings of Smith et al., who demonstrated that TRM cells are maintained in the salivary glands *via* continuous production rather than *via* long-term survival after infection with MCMV (26). In our previous work, we demonstrated that the robust population of M-specific CD8+ TRM cells induced by IN vaccination with MCMV-M contributed to early clearance of RSV (60). This effect was maintained after treatment with a sphingosine 1-phosphate receptor modulator, suggesting that protection was independent of recirculation *via* the lymph nodes. These data concur with the observation herein that IN vaccination with MCMV-M2 failed to mediate early immune control of RSV. Together, these studies highlight the importance of lung-tropic TRM cells in protection against respiratory infection. Accordingly, immunization with a persistent vector offers no immediate advantages over traditional vaccine platforms for conventional epitopes like M2. By contrast, the induction and maintenance of inflationary epitope-specific CD8+ TRM cells in the lungs after vaccination with MCMV may enhance immune protection against respiratory pathogens, which typically induce only transient memory responses at the site of infection (14–16, 24).

Several factors determine the immunogenicity and memory characteristics of any given epitope. In this study, the M and M2 sequences were inserted into the IE2 gene, which naturally encodes inflationary epitopes, and the proteins were under the control of the constitutive promoter IE1 (30, 31, 45). Despite identical genomic locations, the M2 epitope failed to elicit inflationary CD8+ T cell responses. This lack of inflation may reflect greater dependence on the immunoproteasome compared with the M-specific CD8+ T cell response, consistent with previous studies that postulated a key role for antigen processing as a determinant of immunodominance patterns in the context of infection with MCMV (43, 44). In addition, M-specific CD8+ T cells operate with higher composite avidities than M2-specific CD8+ T cells after infection with RSV (57). However, this factor alone may not preclude M2-driven memory inflation, because recent work has demonstrated the existence of low-avidity inflationary CD8+ T cell populations (41). It is also difficult to exclude other possible influences, such as competition between CD8+ T cells with different antigen specificities and variable requirements for co-stimulation and CD4+ T cell help, which are more difficult to assess directly. Any or all of these factors may contribute to the lack of inflation among M2-specific CD8+ T cells. *In vivo* testing is therefore required to assess the true inflationary potential of any given epitope, a process that will become more difficult as vaccines advance from inbred animal models to human populations with diverse genetic backgrounds. A better understanding of the factors that govern memory inflation and how they can be manipulated will be important for the development of CMV vaccines.

As memory inflation is difficult to predict, it is important to study the effect of both inflationary and conventional epitopes in vaccine settings. Coadministration of MCMV-M and MCMV-M2 reduced the overall magnitude of the conventional M2-specific CD8+ T cell response acutely after vaccination but did not impact the inflationary M-specific CD8+ T cell response at any stage after vaccination. Moreover, dual immunization was equivalent to vaccination with MCMV-M alone in terms of protective efficacy after challenge with RSV. These data suggest that both conventional and inflationary epitopes can be included in a persistent vaccine without detrimental effects. However, it should be noted that competition for antigen can occur if inflationary epitopes are delivered by the same vector (45). Individual epitopes are therefore probably best expressed separately if polyvalency is required to prevent immune escape.

In summary, we have shown that memory inflation is required for the maintenance of CD8+ TRM cells in the lungs after IN vaccination with MCMV. These findings highlight an important consideration in the development of persistent vectors and suggest that epitope selection will be a central determinant of efficacy in the setting of vaccines that deliver antigens on a continuous basis.

#### ETHICS STATEMENT

This study was carried out in accordance with the recommendations and guidelines of the NIH Guide to the Care and Use of Laboratory Animals. The protocol was approved by the Animal Care and Use Committee of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Mice were housed in a facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Animal procedures were conducted in strict accordance with all relevant federal and National Institutes of Health guidelines and regulations.

### AUTHOR CONTRIBUTIONS

KM, TR, DP, and BG conceived and designed studies; KM, TR, EB-H, and DN performed animal studies and analyzed data; SM and AR designed and generated recombinant BACs; KM, TR, DP, and BG wrote the manuscript. All authors provided critical feedback and approved the manuscript.

#### FUNDING

This work was supported by the Intramural Research Program of the National Institutes of Health. DP is a Wellcome Trust Senior Investigator (100326/Z/12/Z).

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at https://www.frontiersin.org/articles/10.3389/fimmu.2018.01861/ full#supplementary-material.

# REFERENCES

the growth of mucosal tumors. *Sci Transl Med* (2013) 5:172ra120. doi:10.1126/ scitranslmed.3004888

during murine cytomegalovirus infection. *J Immunol* (2009) 183:3932–41. doi:10.4049/jimmunol.0900227

**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.

*Copyright © 2018 Morabito, Ruckwardt, Bar-Haim, Nair, Moin, Redwood, Price and Graham. 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) and the copyright owner(s) 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.*

*Visai Muruganandah1 , Harindra D. Sathkumara1 , Severine Navarro1,2 and Andreas Kupz1 \**

*1Centre for Biosecurity and Tropical Infectious Diseases, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, QLD, Australia, 2QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia*

Background: Resident memory T cells have emerged as key players in the immune response generated against a number of pathogens. Their ability to take residence in non-lymphoid peripheral tissues allows for the rapid deployment of secondary effector responses at the site of pathogen entry. This ability to provide enhanced regional immunity has gathered much attention, with the generation of resident memory T cells being the goal of many novel vaccines.

#### *Edited by:*

*Fathia Mami-Chouaib, Institut National de la Santé et de la Recherche Médicale (INSERM), France*

#### *Reviewed by:*

*Vaiva Vezys, University of Minnesota Twin Cities, United States Eric Tartour, Hôpital Européen Georges-Pompidou, France*

*\*Correspondence: Andreas Kupz<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 26 March 2018 Accepted: 25 June 2018 Published: 09 July 2018*

#### *Citation:*

*Muruganandah V, Sathkumara HD, Navarro S and Kupz A (2018) A Systematic Review: The Role of Resident Memory T Cells in Infectious Diseases and Their Relevance for Vaccine Development. Front. Immunol. 9:1574. doi: 10.3389/fimmu.2018.01574*

Objectives: This review aimed to systematically analyze published literature investigating the role of resident memory T cells in human infectious diseases. Known effector responses mounted by these cells are summarized and key strategies that are potentially influential in the rational design of resident memory T cell inducing vaccines have also been highlighted.

methods: A Boolean search was applied to Medline, SCOPUS, and Web of Science. Studies that investigated the effector response generated by resident memory T cells and/or evaluated strategies for inducing these cells were included irrespective of published date. Studies must have utilized an established technique for identifying resident memory T cells such as T cell phenotyping.

Results: While over 600 publications were revealed by the search, 147 articles were eligible for inclusion. The reference lists of included articles were also screened for other eligible publications. This resulted in the inclusion of publications that studied resident memory T cells in the context of over 25 human pathogens. The vast majority of studies were conducted in mouse models and demonstrated that resident memory T cells mount protective immune responses.

conclusion: Although the role resident memory T cells play in providing immunity varies depending on the pathogen and anatomical location they resided in, the evidence overall suggests that these cells are vital for the timely and optimal protection against a number of infectious diseases. The induction of resident memory T cells should be further investigated and seriously considered when designing new vaccines.

Keywords: resident memory T cells, infectious diseases, vaccine development, immunity, microbiology

# INTRODUCTION

Traditionally, memory T cells have been subdivided into two broad categories: effector memory and central memory T cells (TEM and TCM, respectively). After the realization that some memory T cells fail to egress out of peripheral tissues back into the blood stream, it became clear that this dichotomous distinction of memory T cells did not account for the complete diversity of the memory T cell population. This led to the discovery of a third subset of memory T cell. Appropriately, dubbed "Tissue-resident memory T cells" (here after referred to as TRM), this newly defined population exhibits the unique feature of remaining localized in peripheral tissues (1). As such, these cells provide enhanced localized immunosurveillance and protection of peripheral tissues when compared to TEM and TCM. TRM have been characterized in many peripheral tissues, including skin, lungs, brain, liver, the female reproductive tract, and the gastrointestinal mucosa. Given the huge variance in their location of residence, this subset of memory cell is highly heterogeneous, phenotypically varying depending on their anatomic location and the inflammatory cues produced by their respective microenvironment. Although experimental techniques such as parabiosis can definitively distinguish TRM from circulating memory T cells, other less complex methods of identifying TRM are more frequently used. The co-expression of CD69 and CD103 is commonly used as a marker of tissue residence, although it appears not all bona fide TRM are defined by this particular phenotype. Regardless, TRM have been implicated in a wide range of physiological functions, such as providing protection against pathogens and cancerous cells, as well as in many pathological states such as autoimmune and other inflammatory diseases. The exploration of TRM biology and the role they play in maintaining homeostasis has broad implications for human health. Currently, our understanding of TRM function is largely constrained within the context of infectious diseases. As of now, it appears that TRM are better adapted to providing rapid protection against pathogen invasion when compared to their circulating counter parts (2–4). Thus, vaccines of the future would ideally establish a population of protective TRM at the portals of entry most at risk of pathogen invasion to provide immediate and effective immunity, rather than relying on the delayed recruitment of effector cells from the circulating pool of memory cells. Since parenterally administered vaccines induce minimal tissuespecific protection, current routes of administering vaccines may need to be revised (5, 6). The present review will primarily focus on the role of TRM in the immune response generated to a range of human pathogens and discuss future avenues for the development of TRM-based vaccines.

# METHODOLOGY

A systematic search of published literature was conducted. Literature was critically evaluated for evidence of the role TRM play during infections and in vaccinology. A flowchart summarizing our methodology has been included (**Figure 1**). The preparation of this review was guided by the *PRISMA-P 2015 guideline* (7).

Figure 1 | Literature search strategy. The search strategy used revealed 381 records in Medline (Ovid), 416 in SCOPUS, and 517 in Web of Science. This resulted in a total number of 1,314 records. After removing duplicates, there were 604 records. Screening of titles resulted in the exclusion of 257 records, as they did not address resident memory T cells, human infectious diseases, or neither. Others records were excluded as they were reviews, editorials, meeting abstracts, book chapters, poster presentations, or erratum notifications. The abstracts of the remaining 347 records were analyzed and a further 124 publications were excluded due to their focus on TRM biology. The full texts of the remaining studies were reviewed. 81 of these texts were excluded for aforementioned reasons. Co-authors were consulted when there was ambiguity regarding the relevance of a study. In total, 142 publications from the search were included. 5 additional studies were included by screening the references of studies from the search results and following external review.

Final searches of literature were performed on March 23, 2018 in Medline, SCOPUS and Web of Science by the first author. The Boolean search strategy used was as following ("resident memory t cell\*" OR "t resident memory cell\*" OR "tissue resident memory cell\*" OR "resident memory" OR "tissue memory"). The references of included studies were also screened for other relevant publications.

Both human and animal studies that use surface markers of residence or other established techniques such as intravascular staining and parabiosis to illustrate localization of T cells to peripheral tissues, as well as T cell phenotyping were included. Studies were also screened for their relevance to human pathogens, and thus animal infection models that are analogous to human infectious diseases were included. Studies were included irrespective of published date. Only published and accepted manuscripts of original research were included. Publications that primarily focused on TRM biology (ontogeny, cellular metabolism, etc.) or non-infectious diseases were not included. Certain non-communicable diseases such as hepatocellular carcinoma and cervical cancer that can be caused by pathogens are briefly mentioned within the broader discussion of TRMmediated immunity.

## RESULTS OF SEARCH

The results of the search strategy are summarized in **Figure 1**.

# DATA SYNTHESIS AND ANALYSIS

The first author conducted extraction of data from relevant studies. This review has been divided into sections based on pathogen type: viruses, bacteria, parasites/helminths, and fungi (**Figure 2**). The studies included in this review contain the most relevant findings related to immune responses generated by TRM against human pathogens, or make use of novel strategies for TRM generation. We apologize to authors whose work could not be included in this review.

diseases (top right), five parasitic diseases (bottom left), and one fungal disease (bottom right). Pathogens have been grouped according to organs or organ systems that have been studied in the context of TRM, including the respiratory, gastrointestinal, and urogenital tracts, as well as brain, skin, liver, lymphatics, and circulation. *Nippostrongylus brasiliensis* and *Heligmosomoides polygyrus* are mouse pathogens for human *Necator americanus* and gastrointestinal helminth infections, respectively. Image modified from https://commons.wikimedia.org/wiki/File:Female\_shadow\_anatomy\_without\_labels.svg.

# THE ROLE OF TRM IN VIRAL INFECTIONS

As of present, TRM immune responses are by far mostly studied in the context of viral pathogens. The following section will present findings by specific viral pathogen/viral disease.

# HERPES SIMPLEX VIRUS (HSV)

Herpes simplex virus causes infections that present with a varying range of symptoms. The primary clinical manifestations of HSV infection are intraepithelial vesicles. There are two antigenically distinct HSV subtypes: HSV-1 and HSV-2, causing cold sores and genital warts, respectively (8). However, both sub-types can be the etiology of either clinical disease as sexual transmission allows for spread between the two sites (9). Both viruses establish a life-long latent infection within the surrounding nervous tissue, and control of HSV infection requires effective cell-mediated immune responses to prevent reactivation. However, co-morbid illnesses, immunosuppressive drugs, UV exposure, and psychological stress can hinder immune control. A number of studies suggest that TRM are implicated in controlling HSV-1 latency in the trigeminal ganglia (10). HSV-1 infection models primarily focus on infections of the skin and nervous tissue such as the trigeminal ganglia and the eyes (11–22). Following acute infection with HSV-1, CD8+ TRM remain localized to the skin initially infected and are also found surrounding latently infected sensory ganglia (11). However, evidence suggests that multiple exposures to cognate antigen can substantially increase the TRM population in not only the site of HSV infection, but also in distant skin (12). CD8+ skin TRM appear to resemble the antigen-presenting Langerhans cells of the skin, extending dendritic projections into the surrounding tissue, probably in an attempt to survey the local area for antigen (13). This is supported by evidence from confocal microscopy and intravital imaging (21) that suggests these TRM can travel between keratinocytes (13). However, unlike Langerhans cells, these TRM do not extend into the stratum corneum (13, 17). It also appears that skin TRM are not specifically attracted to virally infected cells, and thus migrate throughout the epidermis in a random manner (17, 21). By extension, it can be inferred that skin TRM may take a considerable period of time before identifying virally affected cells. As such, it may be safe to assume that a critical mass of skin TRM is needed in order to afford timely protection. This notion is supported by the observation that protection appears to be dependent upon the local density of TRM (17). Upon antigen recognition, skin TRM undergo a change in their morphological and motility pattern, decelerating their migratory rate and losing their dendricity (13, 17). This is probably indicative of a shift in role from immunosurveillance to effector function. Furthermore, the maintenance of HSV-1-specific TRM populations appears to be independent of circulating T cells in both skin and trigeminal ganglia (14, 17). Skin TRM appear to be able to sustain their numbers through local proliferation after secondary infection (17). However, after a combined corticosterone and stress-induced reduction of trigeminal ganglia CD8+ T cells (presumably TRM), there appeared to be no increased proliferation of remaining T cells when compared to the homeostatic proliferation rates as indicated by BrdU incorporation (14). Analysis of HSV-1-specific skin and dorsal root ganglia TRM during acute immunity and later time points revealed that transcription of cytolytic molecules decreases with time. As such, TRM-based immunity in the long term may not be reliant on enhanced cytolytic effector functions, but rather on the localization of these cells at sites susceptible to reinfection (15). The chronic inflammatory response induced by persistent viral gene expression during latency leads to TRM exhaustion in the brain ependymal region, rendering them unable to control HSV-1 infection (16). Perhaps the reason why TRM downregulate their cytolytic genes during times of homeostasis is because continuous expression may lead to exhaustion. Nevertheless, it appears that the generation of CD8+ TRM in skin and ganglia may be a viable option for protection against HSV-1 infection or reactivation. Local inflammation of the skin and mucosa alone can encourage the recruitment of TEM to these peripheral sites where differentiation into the TRM phenotype occurs. This was demonstrated using 2,4-dinitrofluorobenzene, a contact-sensitizing agent. Furthermore, the application of nonoxynol-9 (a spermicide agent) to the female genital tract enhanced protection against HSV challenge, correlating with higher numbers of CD103+ T cells localizing to the epithelium (19). Hence, agents that can be applied to specific tissue and that cause a localized, general inflammatory response may be a strategy worth exploring for the generation of TRM. More specifically, however, the CXCL10/CXCR3 chemokine pathway appears to be vital in generating TRM, as mice deficient in either CXCL10 or CXCR3 were unprotected against HSV-1 UV-B light-induced reactivation challenge. Furthermore, the administration of CXCL10 into deficient mice through the use of a neurotropic virus vector amplified TRM in the trigeminal ganglia, conferring better protections against reactivation challenge (20). CXCL10 administration through the use of a rAAV8-CamKIIa-GFP-CamKIIa-CXCL10 vector showed similar results (18). Samples from human patients that were asymptomatic but seropositive for HSV-1 infection were used to determine which 467 HLA-A\*0201-restircted CD8+ T cell epitopes were immunodominant in the HSV-1-specific immune response. These asymptomatic individuals generated a high number of polyfunctional CD8+ TEM against three epitopes. HLA-A\*0201 transgenic mice were primed with these epitopes and subsequently treated with an ocular topical preparation containing rAAV8-CamKIIa-GFP-CamKIIa-CXCL10 to deliver exogenous CXCL10 chemokine. Results from UV-B reactivation challenge demonstrated that this strategy was able to reduce viral shedding in tears and recurrent herpetic ocular disease (18). This strategy may be beneficial in rationally developing novel vaccines against other diseases.

Human studies have also been conducted in HSV-2 infection. Samples from the genital tract of HSV-2-infected women contained populations of HSV-2-specific T cells with a TRM phenotype (23). More interestingly, a population of CD8αα+ TRM that reside at the dermal–epidermal junction have also been described in biopsies of HSV-2-infected humans. This unique positioning suggests that these cells may be able to survey the neural tissue from which virus travels to the skin during reactivation (24). Thus, TRM play a role in the natural immune response against HSV-2 infection. Although it was already demonstrated that T cells could be recruited to peripheral tissues using inflammatory agents (19), the "prime and pull" vaccine strategy was first described in a HSV-2 infection model (25). In this study, the investigators explored the novel idea of parenterally immunizing mice and subsequently topically administering CXCL10 into the vagina before challenging with HSV-2. Mice that underwent the prime and pull protocol showed minimal signs of clinical disease and had a survival rate of 100%. Naive and parenterally immunized mice that did not receive the pull treatment developed clinical disease and exhibited high mortality rates. This strategy also demonstrated the capacity to prevent infection of sensory neurons (25). Further investigation of this protocol revealed that immunity was largely dependent on INF-γ produced by CD8+ TRM (26). Re-stimulation of this CD8+ TRM was dependent on a population of CD301b+ dendritic cells that resided in the lamina propria. In fact, depletion of CD301b+ dendritic cells using a diphtheria toxin model rendered the prime and pull strategy ineffective and mice suffered high morbidity and mortality rates (26). Although a non-specific inflammatory stimulus such as nonoxynol-9 may be sufficient to pull CD8+ T cells in to the female reproductive tract and subsequently convert them to TRM, it appears that antigen presentation by CD301b+ dendritic cells is needed for CD8+ TRMmediated immunity at this site. A different study that made use of a topical vaccine containing a human papillomavirus (HPV) vector expressing gB and gD ectodomains of HSV-2 has shown the capacity to generate TRM in the reproductive tract, and reduce viral shedding and clinical disease (27). This study highlights the capacity of HPV vectors to induce TRM in the genital tract, a vaccine strategy that may be applicable to other sexually transmitted infections. HSV-2-specific CD8+ TRM can also be generated using a "chemical-free and biological-free" laser adjuvant, a protocol that could be explored in other infectious models (28). While the vast majority of studies have assessed the protective capabilities of CD8+ TRM during HSV infection, very few studies have analyzed the role of CD4+ TRM in HSV infections (21, 29). Intravaginal vaccination of mice with thymidine kinase negative HSV-2 (an attenuated form of the virus) provided full protection against challenge with wild-type HSV-2, independent of CD8+ T cells and B cells. Instead, parabiosis studies demonstrated that CD4+ TRM are required within the genital tract mucosa for immunity in this model. These CD4+ TRM are polyfunctional, secreting IFN-γ, TNF-α, and IL-2, and resided in organized, non-tertiary immune structures called memory lymphocyte clusters (MLCs). These MLCs appear to assemble under the influence of macrophagesecreted CCL5. Upon antigen stimulation, the CD4+ TRM within the MLCs expand and secrete high levels of IFN-γ. The investigators of this study also report that circulating memory T cells were "barely recruited" when MLCs were present in the mucosa. This suggests that CD4+ TRM may be capable of clearing or controlling infection even before recruiting signals are generated in a magnitude large enough to attract circulating T cells to the site of infection (29). It still remains necessary to explore whether a critical mass of CD4+ TRM-containing MLCs are needed within the genital tract to provide protection. It is likely that this profound role of CD4+ TRM in mediating immunity during HSV-2 infection is due to the location of the infection (genital tract) rather than the viral factors alone. This is supported by the fact that MLCs have also been found in the genital tract of both human and mice secondary to infections caused by a bacterial pathogen [refer to *Chlamydia trachomatis* (*Ct*) section of this review]. Despite the difficulties in generating CD4+ TRM following prime and pull vaccination, an ideal vaccine against HSV-2 should generate CD8+ and CD4+ TRM (25).

#### INFLUENZA

Influenza viruses are a major cause of respiratory infections. Although influenza vaccines have been in use for many years, antigenic drift of surface hemagglutinin and neuraminidase proteins require annual immunizations. Antigenic shift can result in highly virulent strains of influenza that cause devastating pandemics (30). The ideal influenza vaccine would provide heterotypic immunity that prevents the escape of newly mutated viruses. Current influenza vaccines rely on generating high neutralizing antibody titers to protect against infection. Although this strategy has demonstrated efficacy in mediating protection, the inability of antibodies to neutralize new variants of the virus has sparked research into alternate strategies (31). Growing evidence suggests that efforts should be focused on developing vaccines that generate TRM-mediated immunity (32). Analysis of human samples has revealed that influenza-specific TRM can be found in substantial numbers in lung tissue, highlighting their role in natural infection (33, 34). Despite expressing low levels of granzyme B and CD107a, these CD8+ TRM had a diverse T cell receptor (TCR) repertoire, high proliferative capacities, and were polyfunctional (34). Influenza infection history suggests a greater level of protection against re-infections likely due to the accumulation of CD8+ TRM in the lungs (35). Furthermore, the natural immune response to influenza A virus infection in a rhesus monkey model demonstrated that a large portion of influenza-specific CD8+ T cells generated in the lungs were phenotypically confirmed as CD69+CD103+ TRM (36). Unlike lung parenchymal TRM, airway CD8+ TRM are poorly cytolytic and participate in early viral replication control by producing a rapid and robust IFN-γ response (37, 38). Bystander CD8+ TRM may also take part in the early immune response to infection through antigen non-specific, NKG2D-mediated immunity (39). The generation of functional TRM that protect against heterosubtypic influenza infection appear to be dependent on signals from CD4+ T cells (40). A role for CD4+ TRM has also been reported (41). Much like their CD8+ counterparts, CD4+ TRM also produce a significant IFN-γ response during early infection (42, 43). Aside from the CD8+ and CD4+ subsets of TRM, a subset of NK1.1+ double negative T memory cells which reside in the lungs also play a role in influenza infection (44). Taken together, these studies and others (45–47) demonstrate that TRM are required for optimal protection. However, unlike TRM in other locations, such as the skin, lung TRM are not maintained for extended periods of time. This gradual loss of lung TRM appears to be the reason for the loss in heterotypic immunity against influenza infection (45, 46, 48). Lung TRM exhibit a transcriptional profile that renders them susceptible to apoptosis (48). Despite conflicting evidence (49), it appears that maintenance of the lung CD8+ TRM populations relies on the continual seeding from circulating CD8+ T cells. However, with time, circulating CD8+ T cells adopt a transcriptional profile that reduces their capacity to differentiate into TRM. Expanding the CD8+ TEM compartment through booster vaccination may circumvent the problem of these time-sensitive transcriptional changes (48). There is also conflicting evidence regarding the requirement of local antigen for the generation of TRM within the lung (48, 50). Continuing to find ways to generate and maintain lung TRM is of great importance for vaccines against pulmonary infections. Intranasal administration of vaccines seems to encourage the development of a strong mucosal immune response (51, 52). Intranasal administration of Live Attenuated Influenza Vaccine (FluMist) in a mouse model induced both CD4+ and CD8+ TRM that provided a degree of cross-strain protection independent of TCM and antibodies (53). The intranasal administration of a PamCys2 or Adjuplex has demonstrated capacity for producing protective influenzaspecific lung CD8+ TRM in similar numbers and IFN-γ secreting potential when compared to the natural response to influenza infection (54, 55). Furthermore, a vaccine containing virus-like particles with tandem repeat M2e epitopes generated heterotypic immunity through the induction of antibodies, and protection correlated with IFN-γ-secreting CD8+ TRM (56). A Modified Vaccinia Ankara-vectored virus expressing conserved influenza nucleoprotein and matrix protein 1 elicited an IFN-γ secreting CD4+ T cell and CD8+ TRM response (57). Co-administration of 4-1BBL (CD137 signal) along with an influenza nucleoprotein expressing replication defective adenovirus vector *via* the intranasal route stimulated and boosted a lung CD8+ TRM response through the recruitment of circulating T cells (58). Intranasal administration of 4-1BBL may serve as a promising "pull" strategy in systemically primed individuals. Another potential "pull" strategy is the intranasal administration of Fc-fused IL-7. This protocol was used as a pre-treatment before influenza A infection, and demonstrated protective capacities in mice against lethal challenge. It appears that Fc-fused IL-7 recruits polyclonal circulating T cells into the lungs, which subsequently reside in the lung tissue as "TRM-like cells" (59). Intranasal administration of Fc-fused IL-7 after systemic priming may be able to recruit influenza-specific T cells into the lungs, and may be a strategy for inducing lung TRM. An antibody targeted vaccination strategy in which antigens are coupled to monoclonal antibodies against CD103+ or DNGR-1+ dendritic cells has also been shown to elicit a protective CD8+ TRM response (47, 60).

 The virus specifically targets the surface proteins CD4, CXCR4, and CCR5, with the natural progression of disease resulting in the depletion of CD4+ T cells. As a consequence, infected individuals are left in an immunocompromised state referred to as aquired immunodeficieny syndrome (AIDS), which is characterized by fatal opportunistic infections and malignancies. Although the development of therapeutics such as anti-retroviral therapy has reduced the incidence of AIDS, HIV/AIDS continues to contribute significantly to global morbidity and mortality. Evidence shows that CD8+ T cells are vital in controlling early infection (61). Studies of human tissue samples have revealed that TRM are generated in response to HIV infection in multiple locations including the gastrointestinal tract and the female reproductive tract (62–65). Furthermore, individuals who appeared to naturally control infection had TRM that were capable of producing the highest polyfunctional immune responses when compared to individuals who did not. However, the TRM population within the HIV-specific CD8+ T cell compartment in individuals who controlled infection was under-represented when compared to individuals who were viremic (62). Although not confirmed, this may be due to the higher ability of polyfunctional TRM in these individuals to recruit circulating T cells, thereby only altering the TRM proportion. Similar to other infections in various sites, CD8+ TRM in the context of HIV can be sub-divided into two subsets based on the expression of CD103 (62, 63). Analysis of the ectocervial epithelium and menstrual blood revealed that HIV-infected women were more likely to have CD103− TRM when compared to healthy individuals (63, 64). This reduced expression of CD103 may be explained by the HIV-induced depletion of CD4+ T cells which appear to be vital in providing help to CD8+ T cells for up-regulating CD103 (64). The CD103− populations of the ectocervix resided closer to the basement membrane of the epithelium when compared to their CD103+ counterparts. Interestingly, the CD103+ population from infected individuals appears to express higher levels of PD-1 (63). In a separate study, adipose PD-1+ CD4+ TRM, appeared to remain relatively inactive during HIV infection and may serve as a reservoir for HIV (65). As such chronically activated TRM and TRM exposed to immunomodulated environments (such as the adipose tissue) may be unable to elicit a full effector response, favoring the progression of HIV infection. It also appears that HIV has the ability to disrupt CCR5 mediated CD8+ T cell migration into the cervical mucosa, thereby impairing the development of TRM populations (66). Regardless, human studies suggest that TRM, especially CD8+ TRM, play an important role in combating HIV infection and thus may be valuable targets for vaccine development. Since the most common mode of transmission of HIV is through sexual intercourse, it may be desirable to explore strategies that induce anti-HIV CD8+ TRM in the female and male reproductive tract and rectosigmoid epithelium. In a Simian Immunodeficiency Virus model of rhesus macaques, intravenous administration of SIVmac239Δ*nef* generated a population of CD8+ TRM in the vaginal tissue and the gut that participated in protection (67). In a murine model, a mucosal vaccination strategy in which intranasal administration of an influenza-vector expressing the HIV-1 Gag protein p24 followed by an intravaginal booster induced CD8+ TRM in the vagina. Antigen stimulation of these CD8+ TRM resulted in the recruitment of B cells, natural killer cells, and CD4+ T cells (68). While the recruitment of innate and adaptive immune cells may be beneficial in early viral clearance, the recruitment of CD4+ T cells may be detrimental in the context of HIV as they are the target for HIV. Hence, incidental recruitment of CD4+ T cells to sites of HIV entry (female reproductive tract and rectum) by prime and pull vaccination strategies may unintentionally increase susceptibility to infection. A micro-needle array delivery system that utilizes a recombinant adenovirus vector containing the HIV-1 protein Gag, has also produced promising results in generating TRM. These HIV-specific TRM were found in the female reproductive tract and respiratory tract of immunized mice and responded to local antigenic stimulation through expansion and production of IFN-γ and granzyme B (69). Using this micro-needle array delivery system as a priming strategy followed by intravaginal delivery of a booster concoction serving as a pull strategy may be an interesting protocol worth exploring.

### VACCINIA

Vaccinia is a poxvirus that usually causes a very mild or asymptomatic infection in immunocompetent individuals. Immunity to vaccinia virus also provides sufficient protection against smallpox, which allowed for its eradication following administration of the live vaccinia virus (70). Despite elimination, smallpox remains a priority on the global agenda given the potential for the virus to be used as a biological weapon (71). For this reason and its ability to serve as a vector, vaccinia virus continues to be used in research. Murine models demonstrate that TRM are generated in response to vaccinia and play a significant role in mediating protection against infection (72–76). Dermal-resident γδ T cells have also been implicated in the immune response against cutaneous vaccinia infection (77). Following skin infection CD8+ T cells are recruited independently of CD4 T+ cells and IFN-γ (72), many of which subsequently assume the TRM phenotype (72, 73, 75, 78, 79) and are capable of initiating potent inflammatory responses upon re-stimulation (79). Of particular interest is the capacity of local vaccinia skin inoculation to globally seed skin tissue even at remote sites with long lasting TRM (72) as well as generating TRM responses in non-related non-lymphoid organs such as the lungs and liver (76). Multiple exposures to cognate viral antigens have also shown to selectively expand TRM (72, 73, 78, 79). In a lung infection model of vaccinia, higher numbers of lung TRM correlated with better protection against subsequent infection as indicated by a rapid reduction in viral loads. TRM seem to expand more rapidly and localize to the infection site as indicated by a 5-ethynyl-2′-deoxyuridine proliferation assay when compared to their circulating counter parts. Depletion of lung CD8+ T cells by intranasal administration of αCD8 antibody, resulted in previously protected mice becoming susceptible to infection, indicating that CD8+ TRM play a vital role in mediating immunity (74). In another study, parabiosis experiments demonstrated that TRM were exceedingly better at clearing vaccinia virus skin infection than TCM within a shorter timeframe. In fact, it appears that skin TRM can clear vaccinia skin infection even in the absence of neutralizing antibodies and TCM (72). However, vaccinia-specific CD8+ skin TRM appear to have an impaired ability to recruit circulating effector cells during polymicrobial sepsis infection (80). Whether there are other physiologically challenging conditions that impair skin TRM functionality remains largely unexplored. Surprisingly, vaccinia lung infection revealed that not all TRM are equally capable of conferring protection. TRM that resided in the lung interstitium were better positioned to rapidly kill infected lung cells in a contact-dependent manner when compared to TRM situated in association with the tissue vasculature. Furthermore, TRM found within the interstitium, unlike vascular-associated TRM, were able to up-regulate CD69 expression, potentially indicating an enhanced ability to respond during early infection (74). Investigations of vaccinia infection has also reinforced that epithelial immunization routes, such as skin scarification and intranasal exposure, demonstrate significant efficacy for generating protective TRM responses (72–76, 78). In fact, vaccination *via* skin scarification is capable of protecting against clinical disease (pock lesions of the skin) whereas not all mice vaccinated *via* systemic routes such as intramuscular and intraperitoneal were protected from pock lesions. More astonishingly, mice immunized *via* skin scarification demonstrated greater resistance to disease when challenged *via* a heterologous route (intranasal), compared to mice immunized subcutaneously or intraperitoneally, in spite of generating reduced antibody titers (75). These observations may be attributed to TRM-mediated immunity given the evidence that TRM can be generated in distant tissues after skin scarification (76). Overall, studies that use vaccinia infection models have shed light on the ability of skin scarification to elicit a robust and somewhat unique immune response.

# RESPIRATORY SYNCYTIAL VIRUS (RSV)

Respiratory syncytial virus is a common cause of lower respiratory tract infections in children and the elderly. Common reinfection with RSV suggests absence of protective immunity (81). A number of studies have shown the importance of TRM in providing protection against RSV (82–86). An experimental human infection study showed that adults with higher frequencies of RSV-specific CD8+ T cells, many of which displayed a TRM phenotype, developed less severe lower respiratory tract symptoms and reduced viral loads. This increase in protection was not correlated with higher numbers of circulating CD8+ T cells, suggesting the localization of TRM was vital for mediating immediate protection (82). TRM induction in lung tissue and airway fluid was also demonstrated following intranasal RSV infection in mice. Adoptive transfer of airway lymphocytes from RSV-infected mice into naïve recipients reduced disease burden upon infection challenge, compared to adoptive transfer of airway lymphocytes from sham-infected mice. It was concluded that both airway CD8+ and CD4+ T cells play a role in protecting against RSV infection and reducing disease severity, respectively (83). However, given that only bulk CD4+ or CD8+ T cells were transferred, it remains to be investigated if the protective capacity is mediated by airway TEM or TRM cells. In support of the latter, an African green monkey model of RSV infection illustrated that antibody and CD4+ T cell responses are unlikely to protect against reinfection. On the contrary, it appears that lung CD8+ T cells, of which up to half displayed a TRM phenotype, were more capable of protecting against secondary infection (84). From the available evidence (82–86), it appears that an ideal RSV vaccine should elicit a CD8+ TRM response in the lung. Of note, some experimental RSV vaccines have already shown promising results with regards to TRM generation: intranasal administration of an RSV antigen-expressing murine cytomegalovirus generated an IFNγand MIP-1β-secreting population of TRM (85); co-administration of the TLR9 agonist CpG and an inhibitor of notch signaling (L-685,458) with formalin-inactivated RSV elicited a strong protective TRM response (86); intranasal administration of virus-like particles containing RSV M and M2 proteins as antigen delivery systems has also shown propensity to induce the production of TRM (87); and a dendritic cell-*Listeria monocytogenes* immunization strategy, when administered locally, was able to avoid circulating T cell-induced immunopathology and protect against RSV infection challenge through the generation of TRM (88).

### CYTOMEGALOVIRUS (CMV)

Cytomegalovirus establishes life-long latency in many organs including mucosal tissues. It has long been known that CMV infection induces a sustained clonal expansion of specific CD8+ T cells, a phenomenon referred to as memory inflation (89). However, only recently has it been explicated that CMV infection promotes the formation of TRM in various mucosal tissues, especially the salivary glands (90–92). Although the CD8+ T cell response is vital for the control of CMV infection, the virusinduced downregulation of MHC I on acinar glandular cells of the salivary glands (long-term target tissue of CMV) resulting in the reliance on CD4+ T cells for control of lytic replication at this site (93). Surprisingly, salivary gland CD8+ TRM were capable of controlling viral replication. It appears that murine CMV is unable to completely inhibit the expression of MHC I on CD8+ TRM of the salivary glands, thereby providing an opportunity for these T cells to mediate localized immunity (90). Although it remains unclear whether these TRM inhibit viral replication through effector cytokines or direct cytotoxicity, it certainly appears that salivary gland TRM may inhibit the shedding of CMV, hence reducing the chances of transmission. These mucosal TRM typically form early after infection. However, mucosal seeding continuously occurs through the recruitment and differentiation of circulating populations. As such, the immunodominance of mucosal TRM against CMV changes with time, favoring the TCR repertoire that remains high in circulation (91). TRM have also been found in brain tissue after murine CMV infection (94–96). In the brain, CMV-specific TRM formation seems to be dependent on regulatory T cell (Treg) activity. Furthermore, Treg cells seem to have a suppressive effect on brain TRM's capacity to produce granzyme B, potentially a precautionary measure to prevent detrimental neuroinflammation (94). From the studies that have dissected the role of TRM in protecting against CMV infection and inhibiting reactivation there seems to be a clear role for these tissue tropic T cells in limiting CMV replication. A number of studies also demonstrate the capacity of CMV to be used as a viral vector in novel vaccines that generate TRM-mediated immunity (85, 91, 97). Manipulating CMV's capacity to induce a robust CD8+ T cell response within mucosal tissues may be a promising avenue for the generation of new vaccines.

## LYMPHOCYTIC CHORIOMENINGITIS VIRUS (LCMV)

Lymphocytic choriomeningitis virus, a rodent-borne disease can cause meningoencephalitis in humans (98). While LCMV infection models have been used to study TRM in multiple tissues (99), the protective role of TRM has only been clearly investigated in the brain, thymus, and female reproductive tract. Depletion of circulating T cells or NK cells demonstrated that TRM have the capacity to protect against infection independently of NK cells, TCM, and TEM populations (100, 101). Upon MHC-I-antigen stimulation, LCMV brain TRM displayed effector functions and mediated virus control through IFN-γ release and perforinmediated cytotoxicity (100). Thymic TRM, when stimulated with gp33, released both IFN-γ and TNF-α, suggesting that TRM at this location may be polyfunctional. It also appears that T cells that took residence in the thymus were more likely to respond to antigen stimulation when compared to their splenic counterparts, further exemplifying the protective nature of these cells (101). Since infection of the thymus can significantly reduce T cell generation due to increased thymocyte deletion and reduced proliferation, it is vital to have protective mechanisms in place that act rapidly to minimize pathogen-induced damage in the thymus. From the available evidence, thymic TRM seem to be capable of adequately fulfilling this task. LCMV infection also induces the production of TRM in various peripheral tissues, such as the lungs, intestines, and female reproductive tract (102–104). While the role of TRM in the lung and intestines following LMCV infection is not well established, it was found that re-activation of CD8+ TRM in the female reproductive tract was able to produce a general anti-viral immune response that is almost able to confer sterilizing immunity when challenged with an non-cognate virus (105). This TRM induced antiviral state may be of great interest in the aim to generate vaccines that create heterotypic protection.

### VARICELLA ZOSTER VIRUS

Varicella zoster virus, the cause of chicken pox, is an alphaherpes virus that can establish latency within the dorsal root ganglia. Reactivation of the virus results in a painful disease called shingles. Although vaccines are available against both chicken pox and shingles (106), recent evidence suggests that TRM may be key players in controlling latent infection, a phenomenon that could be exploited to improve current vaccines. One study analyzed skin samples from human donors of varying ages who were serologically confirmed VZV positive. 80–90% of T cells from the sampled tissue expressed CD69, suggesting that the majority of T cells in skin were TRM. IL-2 responses from stimulated VZV-specific T cells demonstrated that host age did not influence the numbers of responsive cells. However, it was found that skin from older donors demonstrated a lesser capacity to mount a clinical response and Muruganandah et al. TRM in Infectious Diseases and Vaccines

decreased CD4+ T cell infiltration when challenged with VZV antigen. This correlated with higher proportions of Foxp3+ cells. Furthermore, TRM of older skin expressed PD-1 in higher amounts (107). Together, this data suggest that VZV-specific TRM may be suppressed with age. This may be a reason for the high incidence of reactivation of VZV in older individuals. Results from a different study that utilized samples of human trigeminal ganglia suggests that TRM do not seem to play a role in controlling latent infection in the trigeminal ganglia (10). Regardless, further investigation into the role of TRM in controlling latent VZV infection may help to develop therapeutics or vaccines that prevent shingles.

#### HUMAN PAPILLOMAVIRUS

Human papillomavirus is a sexually transmitted pathogen that generally causes an asymptomatic, self-limiting infection. However, certain subtypes of HPV can cause cancer of the cervix, anus, and oropharynx (108). The routine administration of HPV preventative vaccines has led to a significant reduction in the incidence of infection in many parts of the world. However, immunization of individuals with an established HPV infection has not shown to protect against the progression of HPV-induced lesions into carcinoma. As such, a therapeutic vaccine that is administered by post infection may subvert this problem. Current HPV vaccines rely on the induction of antibodies to neutralize viral particles (109). The potential for generating anti-HPV TRM as a strategy for eliminating previously established HPV infection is yet to be fully explored. One study evaluated the capacity of two adenoviruses (Ad26 and Ad35) that express a fusion of the HPV16 oncoproteins E6 and E7 to elicit a protective response in the cervicovaginal mucosa. Intra-vaginal administration of either vector was able to elicit the generation of CD8+ TRM within the cervicovaginal mucosa. Furthermore, systemic priming with Ad35 followed by an intra-vaginal booster immunization of Ad26 induced polyfunctional, E6/E7-specific, cytokine-secreting CD8+ T cells within the cervicovaginal mucosa (110, 111). Although it remains to be resolved if protection against established HPV infection causally relies on TRM, this and other studies (111) provide impetus to further explore the intra-vaginal route of administration and the use of viral vectors as strategies for the induction of cervicovaginal TRM.

### VIRAL HEPATITIS

Viral hepatitis is an inflammatory disease of the liver that is caused by a range of viruses (112). Two studies, both of which utilized human donor liver tissue and paired blood samples, analyzed the role of TRM in the context of viral hepatitis. One study focused on patients with hepatitis B viral infections (HBV), while the other study included patients with HBV or hepatitis C viral infections. A higher proportion of liver T cells from patients who demonstrated partial control of HBV infection had a TRM phenotype, when compared to healthy controls. Given that the overall numbers of T cells in the liver of healthy and HBV-infected individuals were similar, this threefold increase in TRM numbers appear to be due to an increased predisposition of T cells to adopt the TRM phenotype in virally infected liver tissue, rather than expansion of pre-existing TRM (113). The numbers of T cells co-expressing CD69 and CD103 increased by fourfold in chronic hepatitis C patients (114). Furthermore, the reciprocal relationship between viral loads and liver TRM numbers indicates that TRM play a vital role in infection control (113). *Ex vivo* stimulation of TRM showed heterogeneous antigen specificity, with a number of HBV antigens being able to initiate effector responses. However, viral envelope peptides seemed to generate the greatest capacity to induce production of IFNγ, TNFα, and IL-2. Analysis of TRM from healthy liver tissue revealed a noticeably reduced expression of granzyme B, when compared to non-resident counter parts. This suggests that hepatic TRM have less cytolytic capacity than circulating T cells (113, 114). However, liver TRM of patients with chronic hepatitis B expressed markedly higher amounts of granzyme B when compared to healthy controls (114). Liver TRM also showed increased expression of the inhibitory molecule PD-1 compared to non-resident T memory cells (113, 114). The downregulation of granzyme B and upregulation of PD-1 in healthy liver tissue may be a precautionary measure intended to prevent immunopathology, given the liver's role in filtering high amounts of antigen draining from the mesenteric circulation. This is of great importance in viral hepatitis infections as immunopathology is largely involved in the progression of viral hepatitis that leads to cirrhosis and hepatocellular cancer. The increased production of granzyme by TRM in CHB patients may be part of the pathogenesis of fulminant hepatitis. Further exploring the role of TRM in protection against viral hepatitis (including hepatitis A, D, and E) and the immunopathology implicated in the progression of the disease may aid in the development of immunomodulatory therapeutics to prevent viral cirrhosis and hepatocellular cancer.

# EPSTEIN–BARR VIRUS (EBV)