Incomplete Memories: The Natural Suppression of Tissue-Resident Memory CD8 T Cells in the Lung

The yearly, cyclic impact of viruses like influenza on human health and the economy is due to the high rates of mutation of traditional antibody targets, which negate any preexisting humoral immunity. However, the seasonality of influenza infections can equally be attributed to an absent or defective memory CD8 T cell response since the epitopes recognized by these cells are derived from essential virus proteins that mutate infrequently. Experiments in mouse models show that protection from heterologous influenza infection is temporally limited and conferred by a population of tissue-resident memory (TRM) cells residing in the lung and lung airways. TRM are elicited by a diverse set of pathogens penetrating mucosal barriers and broadly identified by extravascular staining and expression of the activation and adhesion molecules CD69 and CD103. Interestingly, lung TRM fail to express these molecules, which could limit tissue retention, resulting in airway expulsion or death with concomitant loss of heterologous protection. Here, we make the case that respiratory infections uniquely evoke a form of natural immunosuppression whereby specific cytokines and cell–cell interactions negatively impact memory cell programming and differentiation. Respiratory memory is not only short-lived but most of the memory cells in the lung parenchyma may not be bona fide TRM. Given the quantity of microbes humans inhale over a lifetime, limiting cellular residence could be a mechanism employed by the respiratory tract to preserve organismal vitality. Therefore, successful efforts to improve respiratory immunity must carefully and selectively breach these inherent tissue barriers.

The yearly, cyclic impact of viruses like influenza on human health and the economy is due to the high rates of mutation of traditional antibody targets, which negate any preexisting humoral immunity. However, the seasonality of influenza infections can equally be attributed to an absent or defective memory CD8 T cell response since the epitopes recognized by these cells are derived from essential virus proteins that mutate infrequently. Experiments in mouse models show that protection from heterologous influenza infection is temporally limited and conferred by a population of tissue-resident memory (TRM) cells residing in the lung and lung airways. TRM are elicited by a diverse set of pathogens penetrating mucosal barriers and broadly identified by extravascular staining and expression of the activation and adhesion molecules CD69 and CD103. Interestingly, lung TRM fail to express these molecules, which could limit tissue retention, resulting in airway expulsion or death with concomitant loss of heterologous protection. Here, we make the case that respiratory infections uniquely evoke a form of natural immunosuppression whereby specific cytokines and cell-cell interactions negatively impact memory cell programming and differentiation. Respiratory memory is not only short-lived but most of the memory cells in the lung parenchyma may not be bona fide TRM. Given the quantity of microbes humans inhale over a lifetime, limiting cellular residence could be a mechanism employed by the respiratory tract to preserve organismal vitality. Therefore, successful efforts to improve respiratory immunity must carefully and selectively breach these inherent tissue barriers.
Keywords: respiratory immunity, influenza infection, cD8 + t cells, cD8 memory, heterologous immunity, tissueresident memory cells iNtrODUctiON Respiratory infections continue to be one of the leading causes of morbidity and mortality worldwide (1). Approximately four million annual outpatient visits are associated with viral respiratory infections, including influenza and respiratory syncytial virus (RSV) (2,3). While a RSV vaccine remains elusive, available influenza vaccines induce specific antiviral neutralizing antibodies that recognize the external antigens hemagglutinin and neuraminidase and are protective against a homologous infection. However, host immune pressure promotes mutations of these antigens between seasons rendering the elicited antibodies and those derived from a natural infection ineffective at providing long-term cross-protection against mismatched or heterologous viral strains (3). Activated CD8 T cells lyse infected lung epithelial cells and produce antiviral cytokines, ultimately eliminating viral reservoirs (4). In the case of influenza infection, CD8 T cells recognize epitopes derived from internal viral proteins that are conserved across 80-100% of circulating influenza strains (4)(5)(6)(7)(8), indicating that elicitation of CD8 T cell immunity could offer a broad range of protection against heterologous influenza infection. This protection would rely on the development of memory CD8 T cells (Tmem) capable of responding rapidly upon challenge (9). However, evidence from murine (6,(10)(11)(12) and human (13,14) studies suggest that long-lived protective Tmem does not form in response to influenza infection. While human studies are lacking, murine models indicate that respiratory anti-influenza Tmem numbers wane coordinate with loss of heterosubtypic immunity to influenza infection (10). This observation, paired with the knowledge that humans are susceptible to seasonal infections following both natural infection and vaccination with the live, attenuated vaccine (3) shows that respiratory Tmem are not stable which we believe is partly due to the incomplete generation of a specific population of Tmem in the lung.

t rM : tHe OtHer MeMOrY ceLL
Infection with various pathogens elicits a heterogeneous Tmem pool that was previously thought to consist of predominately two distinct populations: central memory cells (TCM) located primarily in lymph nodes and effector memory cells (TEM) which circulate through lymphoid and non-lymphoid tissues (15). The preferential localization of TCM is due to expression of CD62L and CCR7 (15), whereas TEM express low levels of these molecules. Tmem develop under a transcriptional program regulated by Eomes (16) and require IL-7 signaling for their survival through T cell contraction (17). However, IL-15 and IL-2 signaling bias Tmem toward a TCM or TEM lineage, respectively (18). In many cases, TEM provide initial pathogen control at portals of entry, while TCM are positioned to broadly patrol lymph nodes (19). Indeed, TCM provide protection against systemic lymphocytic choriomeningitis virus infection (20), while TEM protect against respiratory Sendai virus challenge (21). However, often this is not a true division of labor and, even in the case of non-lymphoid infections, reactivated TCM will also contribute to the generation of new effector cells, albeit with delayed kinetics.
Subsequent studies using parabiotic mice demonstrated the existence of stationary, non-migratory populations of Tmem within the brain and small intestine, and to a lesser extent, other tissues like the lung and liver (22). These cells are now commonly referred to as tissue-resident memory cells (TRM). TRM have a core transcriptional profile that distinguishes them from their TCM and TEM counterparts (23), including expression of transcription factor Hobit (24). How TRM cells developmentally diverge from other Tmem is unclear; however, it is likely to involve early programming followed by acquisition of tissue-specific factors that promote survival and tissue retention (23,25). In most cases, CD8 + TRM have been identified by expression CD69 and CD103 (αE integrin) which are upregulated on TRM in both humans (26,27) and mice (28,29). The ligand of CD103, E-cadherin, is expressed exclusively by epithelial cells and CD69 expression limits tissue egress (30,31), suggesting these markers are responsible for locking TRM within tissues. In fact, TRM fail to develop in the intestines of CD103 −/− mice, and absence of CD69 and CD103 limits TRM formation in the skin (23), indicating that upregulation of CD103 and CD69 are crucial steps for the establishment of TRM. Expression of CD103 and CD69 is regulated by TGF-β (32), which is highly expressed in mucosal sites such as the gut (33) where stable populations of TRM cells have been observed (34). In most cases, TRM are maintained through IL-7-and IL-15-mediated homeostatic proliferation (35,36). TRM are confirmed to exist in the skin (28, 37), brain (38), liver (39), and female reproductive tract (40,41) where they are stably maintained. TRM can persist for up to 120 days in the brain following vesicular stomatitis virus (VSV) infection (38), and skin-resident TRM are the most durable, up to a lifetime in mice following cutaneous herpes simplex virus infection (42).
While a secondary, recall response can be delayed by several days for the activation of Tmem and recruitment of new effectors to the infection site, TRM respond immediately to pathogen reexposure (12). Upon antigen re-encounter, TRM produce IFN-γ (9) to recruit circulating TEM and other immune cells from the blood (43). In addition, TRM can directly kill target cells ex vivo (44), suggesting a cytotoxic potential. TRM have been shown to mediate long-term protection in vivo to infections in the intestine (34), female reproductive tract (40, 41), brain (45), and skin (28,37). Regarding the latter, the smallpox vaccine, administered by skin scarification, generated Tmem which survived for decades (46). While the specific role of TRM in the success of this vaccine is unclear, mice vaccinated via scarification of recombinant vaccinia virus (VacV) generate skin-resident TRM that mediate protection against subsequent VacV infection (47). However, not every infection generates stable Tmem pools. While TRM cells populate the lung and lung airways after influenza infection (12), protection between influenza seasons following natural infection or vaccination with the live-attenuated vaccine is lost (3), suggesting TRM responses may be uniquely regulated in the lung. t rM iN tHe LUNG TRM cells exist within the lung in two distinct compartments: the lung airways and the lung parenchyma. Influenza-specific airway-resident TRM are CD11a lo CXCR3 hi (48,49) and can be isolated by bronchoalveolar lavage. It is estimated that antiinfluenza TRM in the lung airways have a half-life of only 14 days, and for some period of time are continually replenished from the circulating TEM pool (48). Interestingly, airway TRM have a low cytolytic capacity and fail to proliferate upon antigen re-encounter but rapidly produce antiviral cytokines such as IFN-γ (44). TRM embedded in the lung parenchyma are CD11a hi CXCR3 lo , highly cytolytic and undergo rapid proliferation after antigen re-exposure (44). We have known for some time that regional Tmem are responsible for limited heterologous immunity after respiratory infection (10). A careful study of the kinetics of Tmem decay after Sendai and influenza virus infections demonstrated a rapid decline in Tmem numbers in the lung and lung airways by 90 days postinfection. Importantly, this loss of influenza-specific Tmem in the lung coincided with loss of heterosubtypic immunity to influenza infection (10). The attrition of influenza-specific cells is restricted to the lung, as splenic memory cell numbers do not decline, indicating this is likely loss of the TEM or TRM pools. Subsequent experiments demonstrated that airway CD103 + cells are responsible for protection against a secondary, heterologous virus challenge. However, this pool declines rapidly after infection and is undetectable within 7 months postinfection (12), in part due to the inhospitable environment of the lung airways. TRM in the airways reside at the frontline, adjacent to influenzasusceptible epithelial cells. However, lung parenchymal TRM and circulating TEM are also available within the lung tissue and can serve as a secondary line of defense. Recent evidence indicates that over time, TRM cells in the lung airways wane and are replaced by circulating TEM cells; however, these TEM also decline and lose the ability to convert to TRM (50). This, coupled with a loss of TRM in the lung parenchyma, results in a gradual decline in the overall TRM population in the lung. Decline in the lung parenchymal TRM pool could be due to increased cell death, limited proliferation, or emigration. Unlike TRM in other sites (28,34,38), most lung Tmem do not undergo homeostatic proliferation (50,51). However, a small pool is replenished from proliferating Tmem that have recently emigrated from secondary lymphoid tissues (50). In addition, there is no evidence that TRM cells in the airways egress from the lung or re-enter circulation (48). Therefore, we propose that Tmem embedded in the lung tissue are either eventually lost to the airways or do not represent a bona fide, protective TRM pool. Our opinion that lung parenchymal TRM do not exist is based on two observations. The first is that few Tmem truly penetrate into the tissue and the second is that those Tmem that do, are not CD103 + CD69 + .
Many techniques can identify TRM ( Table 1) and each has pros and cons. We believe that the most effective methodology is the combination of two of these approaches: intravascular staining and CD103/69 phenotyping. Intravascular staining distinguishes between cells circulating through the blood and those embedded within a tissue (52). Approximately 99% of the TRM within the epithelial layer of the small intestine are protected from the intravascular staining (Figure 1) (52,53), validating similar results observed in parabiotic mice (22). In contrast, the majority of the memory cells within the lung parenchyma 35 days after respiratory infection with either influenza, VSV, or Listeria monocytogenes are part of the circulating TEM pool, with only 10-20% of the cells in the lung parenchyma truly within the tissue (52) (Figure 1). These data do contrast with other respiratory infections that are skewed toward the upper respiratory tract (54) or are chronic (55), both cases generating CD103 + CD69 + TRM. With regard to the latter study, it is possible that persistent antigen and inflammation is required for the successful development of TRM within this site. In addition to antigen access, antigen competition can regulate TRM populations at the clonal level (56). Moreover, many studies identify lung TRM via CD103 and CD69 expression on isolated lymphocytes (57,58), independent of intravascular staining. However, expression of these markers does not always correlate with tissue residency. For example, some TRM cells in the lamina propria of the gut (59), the liver (39), and the brain (60) are CD103 − , and human splenic Tmem can be CD69 + (26). In fact, less than 30% of the IV protected TRM cells isolated from the lung  parenchyma express CD69 and CD103 (Figure 1) compared to TRM isolated from other mucosal sites, where expression ranges from approximately 50-99% (59). Therefore, Tmem located in the lung parenchyma after respiratory infection lack one of the key attributes associated with bona fide TRM, expression of CD69 and CD103. CD103 − TRM in the brain are maintained for a few months (60) which may be due to modified tissue localization and enhanced access to IL-15. However, lung parenchymal TRM are maintained independent of IL-15 (61), at least in the short-term, so gained proximity to IL-15 may not matter. However, acquisition of other survival signals dependent on CD103 positioning may be altered, leading to either cell death or assimilation into the TEM pool. Coupled with loss of airway-associated TRM, this situation leaves circulating TEM as the only viable responders. Whether the TEM temporally supplementing the TRM pool are CX3CR1 hi and classified as the recently described "peripheral" memory cells (TpM) (62, 63) is unknown. Nonetheless, as TEM induced from respiratory infection decline over time (64), hosts will then be susceptible to infection. Therefore, an inferior CD69 + CD103 + TRM response underpins loss of heterosubtypic immunity in the lung and raises the question of why long-lived, stable TRM does not form in the lung following respiratory infection.

tHe resPiratOrY eNvirONMeNt sUbverts tHe DeveLOPMeNt OF t rM
As the lung is exposed to both infectious agents and innocuous environmental antigens, immune responses must be tightly controlled to prevent immunopathology (25). Similar regulation is also required in the liver and brain, additional tolerogenic sites. In part, this regulation is accomplished via tissue segregation. Indeed, liver TRM are exclusively segregated from tissue stroma, retained within the sinusoids (74), whereas brain TRM are preferentially localized in the meninges and perivascular areas (60), sequestered from the parenchyma. The lung is no different, with the development of BAL TRM and parenchymal TRM. However, unlike TRM in the brain and liver, BAL TRM are directly exposed to the external environment and easily lost, whereas the lung parenchymal TRM are imbedded in the parenchyma and require an additional level of regulation to prevent immunopathology. One potential mechanism is through altered mammalian target of rapamycin (mTOR) signaling within the respiratory tract. mTOR is responsible for regulating cellular metabolism, proliferation, and differentiation (75), including memory cell development (76). High levels of mTOR activation reduces the total number of antigen-specific cells expressing CD127, required for the development of memory precursor cells (77), and the subsequent TCM pool (76). While reducing mTOR signaling with rapamycin reverses the effects on TCM (76), TRM formation and retention within the intestinal mucosa was also increased via enhanced expression of gut-specific homing molecules (78). To date, no study has linked reduced mTOR signaling to enhance lung homing and/or respiratory TRM formation. However, evidence from viral respiratory infection models support a role for mTOR in TRM formation. Rapamycin treatment during influenza infection increases the total number of antigen-specific CD8 Tmem circulating in the blood (79) similar to studies in the gut (78). In addition, activated CD8 T cells isolated from infants infected with RSV and treated with rapamycin during in vitro re-stimulation express higher levels of CD127 compared to those cells stimulated without rapamycin. Rapamycin treatment also enhanced the effector response of RSV-specific cells by increasing their proliferation and production of granzyme B (80). While increased infiltration of RSV-specific effector cells into the lung may be important for viral clearance, this can also result in damaging pathology within the lung tissue itself. This indicates that perhaps careful regulation of mTOR signaling during respiratory infection is important for limiting potential immunopathology (80) and Tmem development; however, further studies are needed to directly implicate mTOR as a player in lung TRM formation.
The lung environment is inherently immunosuppressive. In the steady state, a large reservoir of Tregs populate this tissue and contribute to significant IL-10 post-influenza infection (81). Moreover, bronchial and alveolar epithelial cells are known to express moderate levels of the programmed death-1 (PD-1) ligands PD-L1 and PD-L2, both of which are significantly upregulated upon RSV (82) and influenza infection (83). In addition, antigen-specific CD8 T cells infiltrating the lung following RSV and influenza infection have an increased expression of PD-1 (83,84). Both IL-10 and PD-1 signaling can modulate CD8 T cell activation both individually (85,86) and cooperatively (87) by tuning TCR signaling. IL-10 suppresses IL-12 signaling which, like PD-1 signaling, activates mTOR. However, PD-1 signaling is not exclusively through mTOR and can affect transcriptional networks and other cell cycle regulators which can impact the fate and function of CD8 + T cells (86). Memory phenotype cells isolated from PD-1 −/− versus wild-type mice are preferentially TEM (88). Reciprocal adoptive transfer experiments demonstrated this bias was inherent to the T cell. As PD-1 blockade during RSV infection results in enhanced inflammation and lung injury, PD-1/ PD-L1 expression in the respiratory tract may serve to limit the expanding CD8 + T cell pool, thereby restricting developing TRM. Thus, while enhanced PD-1 expression within the respiratory tract may be important for regulating inflammation, this may create an environment that is inhospitable to the formation of TRM.
It is also possible that respiratory infections alter TRM programming via inhibition of CD103 and CD69 expression, which negatively affects the formation and/or retention of TRM cells in the respiratory tract. Constitutive expression of TGF-β in mucosal sites such as the gut (33) is crucial for the development of longlived TRM through induction of CD103 expression (89). Epithelial cells also provide survival signals such as IL-15 (90), thus high CD103 expression may not only facilitate TRM retention but aid in their development or survival via tissue positioning. However, high levels of TGF-β in the respiratory tract can be detrimental, leading to the development of cystic fibrosis within the lung (91). Although TGF-β expression is induced by influenza infection (92,93), it may only be transiently expressed to limit immunopathology, albeit at the expense of TRM formation. In fact, the TRM in peripheral sites can cause semi-permanent scarring in tissues that worsens after TRM re-activation and production of IFN-γ in situ (94). Since high levels of IFN-γ production (95), in addition to scarring and fibrosis in the lung, can cause respiratory failure (96), the retention of TRM long term may be inherently limited to maintain host fitness. If this is the case, promoting TRM formation within the respiratory tract could have severe consequences for host respiratory health. Therefore, by reducing TGF-β, and coordinately CD103 expression, lung memory precursor cells would perhaps be ill positioned to receive homeostatic signals responsible for the development, survival, and/or retention of TRM and could be either be lost or assimilated into the TEM pool.
While airway-resident TRM cells confer protection against secondary influenza infection, they rapidly wane, leaving only parenchyma resident TRM and circulating TEM to maintain protection against subsequent infection. However, TEM also wane over time (64) and the formation of bona fide TRM in the lung parenchyma is limited (Figure 1). These incomplete memories leave the host susceptible to recurring influenza infection. We believe the lung evokes a form of natural immunosuppression whereby inhibitory signals in the site protect the host from debilitating tissue damage while simultaneously suppressing the formation of bona fide TRM within the lung tissue. While the exact mechanisms that underlie altered TRM formation within the respiratory tract are still not fully understood, future efforts to improve the maintenance and stability of this population must bear caution due to potentially negative, long-term effects on the host. Moreover, in developing vaccines against respiratory pathogens, it will be important to identify strategies that will prevent re-infection with respiratory viruses without compromising host respiratory health.

etHics stateMeNt
All animal studies were conducted under guidelines approved by the Institutional Animal Care and Use Committee of the University of Georgia.

aUtHOr cONtribUtiONs
Both KK and KR conceived and wrote the perspective.
acKNOWLeDGMeNts Special thanks to Dr. Hillary Shane for critical reading of the manuscript.

FUNDiNG
This work was sponsored by NIH grant AI131093 to KDK.