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Airway resident memory CD8 T (TRM) cells are a distinctive TRM population with a high turnover rate and a unique phenotype influenced by their localization within the airways. Their role in mediating protective immunity to respiratory pathogens, while suggested by many studies, has not been directly proven. This study provides definitive evidence that airway CD8 TRM cells are sufficient to mediate protection against respiratory virus challenge. Despite being poorly cytolytic in vivo and failing to expand after encountering antigen, airway CD8 TRM cells rapidly express effector cytokines, with IFN-γ being produced most robustly. Notably, established airway CD8 TRM cells possess the ability to produce IFN-γ faster than systemic effector memory CD8 T cells. Furthermore, naïve mice receiving intratracheal transfer of airway CD8 TRM cells lacking the ability to produce IFN-γ were less effective at controlling pathogen load upon heterologous challenge. This direct evidence of airway CD8 TRM cell-mediated protection demonstrates the importance of these cells as a first line of defense for optimal immunity against respiratory pathogens and suggests they should be considered in the development of future cell-mediated vaccines.
Clearance of a primary respiratory virus infection results in the establishment of virus-specific central memory T (TCM) cells that reside in secondary lymphoid organs, effector memory T (TEM) cells that recirculate through tissues, and resident memory T (TRM) cells that remain in the lung parenchyma and lung airways (1). At the population level, both airway and parenchymal TRM cells display similar kinetics where the number of antigen-specific memory CD8 T cells is highest in these sites at one month post-infection and gradually declines before stabilizing at a relatively low number of cells six to eight months post-infection (2). However, the homeostasis of these populations at the level of individual cells is quite different. Whereas lung parenchymal TRM cells are long-lived in the tissue, airway TRM cells have a relatively short half-life of approximately 14 days and must be continually replenished to maintain the population (3). Thus, even though these resident memory populations occupy the same tissue, the differences between them at the level of individual cells make it unclear whether they equally contribute to cellular immunity in the lung.
Memory CD8 T cells canonically aid in controlling and clearing a pathogen through targeted lysis of infected cells (4) and modulation of the innate immune response at the site of infection through the local production of cytokines (5). There is ample evidence from animal models that memory CD8 T cells confer protective immunity to respiratory viruses by significantly decreasing viral loads, leading to faster clearance and decreased immunopathology (6–8). Recent studies in humans showed that increased numbers of circulating cross-reactive memory CD8 T cells correlated with significant decreases in viral loads and lower disease burden following heterosubtypic influenza challenge (9). Notably, studies in animal models that allow sampling of peripheral tissues have shown the number of memory CD8 T cells in the lung correlates with the efficacy of cellular immunity to respiratory virus challenge, and a similar phenomenon has been observed in models of M. tuberculosis immunity (10, 11). Furthermore, the protective efficacy of cellular immunity to influenza virus slowly declines over several months post-infection with kinetics identical to the decline in the number of airway CD8 TRM cells (12). Previous studies have shown that airway CD4 TRM cells could mediate protection in mice lacking CD8 T cells (13), but despite the potential correlation between airway CD8 TRM cells and protective cellular immunity in the lung, there is currently no direct evidence that demonstrates the protective efficacy or protective mechanism of these cells.
TRM cells are generated in response to regional infections and have been documented in the lungs, skin, gut, and reproductive tract where they would have the ability to provide an initial line of defense against invading pathogens (14–19). TRM populations consist of non-circulating cells characterized by permanent residence in peripheral tissues; expression of the tissue retention molecules CD69 and CD103; down-regulated expression of CD62L, CCR7, and sphingosine-1-phosphate receptor 1 (S1PR1); and a transcription program distinct from their circulating TEM cell counterparts (20, 21). Despite sharing these hallmarks with TRM populations in other tissues, lung airway TRM cells have a distinct phenotype and are short-lived, likely due to the harsh airway microenvironment. Key features of this distinct phenotype are the down-regulation of the integrin CD11a and poor in vivo cytolytic capacity, which call into question the ability of these cells to participate in protective immunity (22, 23) Nevertheless, airway CD8 TRM cells are in prime position to respond to a challenge from pathogens that infect the respiratory epithelium (24). Therefore, it is important to know whether these cells are sufficient to protect against secondary challenge and if so, how they mediate said protection.
In this study, we use an intratracheal transfer approach to show that airway CD8 TRM cells are sufficient to convey protection against respiratory virus challenge in an antigen-specific manner and quickly produce IFN-γ upon antigen exposure to limit early viral replication in the lung. We used murine models of influenza and Sendai virus infection to demonstrate that airway CD8 TRM cells are equally sensitive to antigen as spleen-derived TEM cells; however, airway CD8 TRM cells respond more quickly, with the predominant responsive population being long-term airway resident cells rather than cells having recently migrated from the lung parenchyma or vasculature. Finally, we show that transfer of airway CD8 TRM cells lacking IFN-γ have a significant defect in their protective efficacy. Our findings on the protective capacity of airway CD8 TRM cells demonstrate their utility in providing protective immunity against respiratory pathogens, lending insight into a protective cellular population that could be elicited through future targeted cellular-based vaccines or immunotherapies.
C57BL/6J (WT), B6.PL-Thy1a/CyJ (CD90.1), B6.SJL-Ptprca Pepcb/BoyJ (CD45.1) and B6.129S7-Ifngtm1Ts/J (IFN-γ KO) mice from The Jackson Laboratory were housed under specific ABSL2 conditions at Emory University and Trudeau Institute. Intranasal infection with influenza A/HKx31 (H3N2) at 30,000 50% egg infectious doses (EID50) and Sendai virus at 282 EID50 established virus-specific T cells in mice as previously described (25). Influenza A/PR8 (H1N1) at 6,000 EID50 was used for challenge of transfer recipient mice. All experiments were completed in accordance with the Institutional Animal Care and Use Committee guidelines of Emory University and Trudeau Institute.
Memory CD8 T cells, harvested from mice 35–45 days post-infection, were negatively selected from bronchoalveolar lavage (BAL) using Miltenyi CD8α T Cell Isolation Kit II. Influenza NP366–374/Db+ tetramer quantification allowed for equal numbers of antigen-specific cells to be i.t. transferred from donor mice to naïve recipient mice. No more than 1.5×105 antigen-specific airway CD8 TRM cells were transferred per recipient to approximate physiological numbers of airway TRM cells. Antibodies used for flow cytometry and cell sorting were BioLegend CD62L [MEL-14], CD8α [53–6.7], CXCR3 [CXCR-173]; eBioscience CD11a [M17/4], CD44 [IM7]; and BD Biosciences CD3ε [145-2C11], CD45.2 , CD90.2 [53–2.1], IFN-γ [XMG1.2]. Intravital staining was performed immediately before mouse euthanasia and tissue harvest as previously described (15). Briefly, to identify T cells resident in various tissues, including the lung parenchyma, 1.5µg of fluorophore-conjugated α-CD3ε antibody in 200λ 1× PBS was intravenously injected into the tail vein of mice; five minutes post-injection, mice were euthanized with Avertin (2,2,2-Tribromoethanol - Sigma) and exsanguinated prior to harvest of BAL and other tissues. Staining for intracellular cytokines was performed as previously described following stimulation in the presence of Brefeldin A for the indicated periods of time (25). To study cell proliferation, mice were given an intraperitoneal bolus of BrdU (0.8mg) at the time of infection and maintained on BrdU drinking water (0.8mg/mL) until harvest. BrdU incorporation was measured using the BrdU Flow kit (BD Biosciences) following tetramer and antibody staining. Samples were run on a BD Biosciences Canto II or LSR II flow cytometer and analyzed with FlowJo software. Sorting was performed on an Influx or Aria II cell sorter (BD Biosciences).
Donor airway CD8 TRM cells were harvested from the airways of PBS control or PR8 challenged mice and sorted based on CD90.2 expression. Congenic (CD45.1+) targets were pulsed with FluNP366–374 peptide (specific targets) or a non-specific peptide (γHV p79524–531 or SendaiNP324–332) for two to four hours at 37°C with non-specific targets being labeled with 2.5µM CFSE; non-specific and FluNP targets were then mixed at a 1:1 ratio. Sorted airway CD8 TRM, lung parenchymal CD8 TRM, or splenic CD8 TEM cells were incubated with targets at an effector to target (E:T) ratio of 4:1 or 1:1 for six, 14 or 24 hours. The ratio of live specific targets to live non-specific targets was determined by gating on propidium iodide−/CD45.1+/CFSE+/− cells following flow cytometry. Specific lysis was calculated by the formula:.
Mice who received PBS, Sendai-specific airway CD8 TRM cells, or influenza-specific airway CD8 TRM cells followed by intranasal influenza virus challenge the following day had BAL isolated three days post-challenge. The supernatant from the single BAL pull was separated from cells via centrifugation prior to cytokine and chemokine analysis by Luminex. Alternatively, BAL and spleens were harvested from Sendai memory mice, sorted to isolate CD44hi/CD62L−/CD8+ cells, and were stimulated 6 hours using irradiated congenic APCs pulsed with 1µg/mL SendaiNP324–332 (FAPGNYPAL) or 1µg/mL FluNP366–374 (ASNENMETM) prior to cytokine and chemokine analysis by Luminex.
Sendai and influenza virus PFU titers were completed as previously described (7) following day three post-challenge with Sendai or ×31 influenza virus, respectively. Quantitative PCR on influenza virus polymerase gene (PA) was completed as described (26), using High Capacity cDNA Reverse Transcription Kit (Life technologies) generated cDNA from 2µg RNA isolated from lung homogenates by TRIzol and RiboPure RNA Purification Kit (Ambion).
Given their proximity to the respiratory epithelium, airway TRM cells are ideally located to rapidly recognize and respond to respiratory viral infections. However, prior ex vivo studies have shown airway CD8 TRM cells have a unique phenotype and effector function when compared to their systemic counterparts. Because of these differences, it is unclear if, and in what capacity, these cells contribute to protective immunity. To specifically test the protective capacity of airway CD8 TRM cells in the absence of parenchymal TRM and circulating TEM cells, we intratracheally (i.t.) transferred Sendai or influenza virus-specific airway CD8 TRM cells from the airways of immune mice directly into the airways of naïve recipient mice (Figure 1A). The transferred airway CD8 TRM population expressed high levels of CXCR3 (Supplemental Figure 1), which has been shown to be up-regulated on CD8 T cells in the airways during an acute infection and continues to be expressed into immunological memory (27). These cells also remain in the airways following i.t. transfer and do not egress from the airways to the lung parenchyma or mediastinal lymph node (MLN) (Supplemental Figure 2). Recipient mice were challenged with influenza or Sendai virus one day after transfer and viral titers were measured three days after infection. As shown in Figures 1B and 1C, mice receiving airway CD8 TRM cells specific to the challenge virus had a significant decrease in viral titers. In contrast, airway TRM cells specific for a different virus showed no difference in titers compared to PBS controls. Thus, airway CD8 TRM cells are sufficient to limit early viral replication through a mechanism that requires cognate antigen recognition.
Three days after intranasal challenge, the BAL supernatant was harvested from naïve mice who received an i.t. transfer of PBS, Sendai-specific airway CD8 TRM cells, or influenza-specific airway CD8 TRM cells one day before challenge (Figure 2A). Despite having the greatest reduction in viral titers upon challenge, mice receiving influenza-specific airway CD8 TRM cells i.t. produced significantly lower levels of CXCL-1, CCL-2, IL-6 and TNF-α when compared to mice receiving PBS or Sendai-specific airway CD8 TRM cells (Figure 2B). In contrast, the airways of naïve mice receiving Sendai-specific airway CD8 TRM cells i.t. had higher levels of all four inflammatory cytokines than even the PBS controls as a result of the non-antigen-specific influenza virus challenge. Therefore, upon exposure to cognate antigen, the ability of airway CD8 TRM cells to rapidly decrease viral loads aids in restraining the local pro-inflammatory immune response and limiting unnecessary damage to the lungs.
To understand the mechanism by which airway CD8 TRM cells mediate protection, we examined the capacity of these cells to induce target cell death in vitro and their ability to proliferate upon secondary infection. In Figure 3A, we isolated and sorted airway CD8 TRM and splenic CD8 TEM cells from ×31 influenza memory mice to compare their respective cytolytic capabilities. The specific lysis of airway CD8 TRM cells was relatively negligible, remaining at ~10%, for E:T (effector to target) ratios ranging from 1:1 to 4:1, while the specific lysis of splenic CD8 TEM cells from the same mice increase as the E:T ratio increases (Figure 3A). For all three E:T ratios, the specific lysis of the splenic CD8 TEM cells was significantly higher than that of the airway CD8 TRM cells. To directly compare the CTL activity of the airway (BAL) and lung parenchymal (LP) CD8 TRM cells 35 days post-x31 influenza virus infection, we sorted cells from the airways and lung tissue which were protected from an intravital staining antibody, providing a CD44hi/CD62Llo CD8 TRM population from each resident compartment (Figure 3B). Figure 3C shows that, at a 1:1 E:T ratio, the LP CD8 TRM cells have significantly higher CTL activity than the airway CD8 TRM cells, even after incubating with targets for 14 hours. Finally, to understand if the airway CD8 TRM population gains CTL function by encountering cognate antigen, we transferred airway CD8 TRM cells from ×31 influenza-primed mice i.t. into congenic naive mice and challenged those mice with PBS (control) or PR8 influenza (PR8). On day three post-challenge, the transferred airway CD8 TRM cells were isolated by cell sorting and assessed for cytolytic function. Even in the presence of cognate antigen stimulation in vivo, airway CD8 TRM cells remained poorly cytolytic in a short-term CTL assay irrespective of the E:T ratio and did not display robust cytolytic function until 24 hours of target incubation (Figure 3D). Therefore, the airway CD8 TRM population, once established, is poorly cytolytic and remains poorly cytolytic even during a secondary infection, while the lung parenchymal CD8 TRM population retains their cytolytic capacity.
To investigate whether the rapid proliferation and expansion of airway CD8 TRM cells may be important for their protective function, we transferred airway CD8 TRM cells (CD90.2+) from ×31 influenza-primed mice i.t. into congenic (CD90.1+) ×31 influenza-primed mice, challenged with PR8 the following day, and maintained the mice on BrdU water for seven days (Figure 4A). The i.t. transferred population maintained their CD11alo status, did not incorporate BrdU, and failed to expand throughout the secondary response (Figure 4B and 4C). Notably, the only flu-specific CD8 T cells in the airways to incorporate BrdU were host cells that recently migrated to the airways, as noted by their CD11ahi status; these host cells eventually dominate the secondary response. Together, these data infer that the airway CD8 TRM cells do not need to proliferate within the airways or gain rapid cytolytic function to mediate protection to a secondary challenge.
Given their suboptimal cytolytic activity, we hypothesized that airway CD8 TRM cells may provide protection by rapidly detecting cognate antigen and secreting antiviral cytokines in response to secondary challenge. To test this, we examined the cytokine profile of airway CD8 TRM cells and splenic-derived CD8 TEM cells from Sendai-immune mice in response to their cognate antigen (SendNP) or an unrelated peptide (FluNP). As shown in Figure 5A, after six hours of stimulation with cognate antigen, airway CD8 TRM cells produced significant amounts of IFN-γ, TNF-α, and IL-10; splenic CD8 TEM cells also produced significant amounts of these cytokines plus IL-2. Notably, out of the cytokines produced by airway CD8 TRM cells, IFN-γ was most impressive with respect to magnitude and merited further investigation.
We suspected that the rate at which the airway CD8 TRM population senses its cognate antigen could be another difference between the two populations, as rapid cytokine production is a hallmark of TRM-mediated protection in other peripheral sites (18). This would corroborate the idea that the airway CD8 TRM population acts as an early warning sensor to mediate protection in an antigen-specific manner. Thus, when we compared the airway CD8 TRM and splenic TEM cell IFN-γ production at early times after cognate peptide stimulation (Figure 5B), we observed that the airway CD8 TRM population reacted faster (within two hours) than the splenic CD8 TEM population. Furthermore, it was the CD11alo airway CD8 TRM population, which has resided in the airway the longest, that had the fastest rate of IFN-γ production. One explanation for the quicker IFN-γ response by the airway CD8 TRM cells is that they are more sensitive to antigen than splenic CD8 TEM cells. However, there was no difference in peptide affinity between airway CD8 TRM and splenic CD8 TEM cells (Figure 5C). This lack of difference is especially true at lower concentrations where a divergence would be expected if the airway CD8 TRM cells had greater functional avidity to their cognate antigen than the splenic CD8 TEM cells. Together, these data demonstrate that airway CD8 TRM are able to rapidly produce antiviral cytokines upon antigen recognition and suggest that airway CD8 TRM cell-derived IFN-γ may be a crucial mediator of protection against respiratory virus challenge.
To test if IFN-γ was important for airway CD8 TRM cell-mediated protection during an influenza virus infection, we i.t. transferred equal numbers of FluNP-specific airway CD8 TRM cells from either WT or IFN-γ-deficient mice into naïve recipients, followed by PR8 influenza challenge one day later (Figure 6A). We found that mice receiving influenza-specific WT airway CD8 TRM cells have significantly lower viral copies than those mice receiving airway CD8 TRM cells from IFN-γ-deficient mice following PR8 challenge (Figure 6B). Moreover, mice receiving IFN-γ-deficient airway CD8 TRM cells still showed a significant decrease in virus copies compared to PBS control mice, suggesting that other antiviral mechanisms are likely involved, such as production of TNF-α by airway CD8 TRM cells observed in Figure 5A. Nevertheless, while it has been shown that IFN-γ is not necessary to survive a lethal primary influenza virus infection (28), these data show that IFN-γ produced by airway CD8 TRM cells plays an important role in limiting viral loads following secondary challenge, which can be important in limiting immunopathology during an infection (29).
Tissue-resident memory T cells established at thresholds of pathogen entry play a crucial role in protective immunity. These findings provide the first direct evidence that airway CD8 TRM cells, a unique population of TRM cells based on their limited lifespan and microenvironment-constrained phenotype, serve as a first line of defense in the lung against pathogen challenge and are sufficient to limit early viral replication. Their fast response upon antigen exposure to produce IFN-γ and other effector cytokines makes them ideal for limiting early viral replication. Furthermore, these cells fail to proliferate within the airways and remain poorly cytolytic even in the presence of their cognate antigen, suggesting that the ability to rapidly produce cytokines is critical for their protective efficacy. In support of this, airway CD8 TRM cells lacking IFN-γ had a significant defect in protective immunity compared to wild-type controls. Together, these data demonstrate that the airway CD8 TRM population plays an important role in secondary cellular immunity against respiratory viruses by providing a rapid, local source of cytokines to promote an early anti-viral state.
Many studies have observed a correlation between the steady decline in numbers of airway CD8 TRM cells in the months after primary infection and the steady decline in heterosubtypic immunity against influenza virus challenge. However, demonstrating that the decline in protective immunity is a direct consequence of a decline in airway CD8 TRM population has been difficult because delineating the individual contributions of airway TRM, lung parenchymal TRM, and circulating TEM populations are not possible through traditional antibody depletion approaches. The importance of analyzing the role of airway CD8 TRM cells independently of these other subsets was further highlighted in a recent study that observed that lung parenchymal TRM cell numbers also decline in the months post-infection, and the decline in protection may have been solely attributable to this phenomenon (10). Our data do not preclude a role for parenchymal TRM cells in heterologous immunity, but rather suggest that these populations may act in concert to limit early viral replication. Unlike the airway TRM population, TRM populations within other tissues display strong cytolytic activity, and the lung parenchymal TRM population maintains expression of CD11a, enabling their cytolytic activity (Figure 3C). It has been shown that infected lung epithelial cells can present antigen to T cells on the apical surface lining the airways in addition to the basolateral surface; so, it is possible that an infected epithelial cell would be presenting antigen to both the airway and lung parenchymal TRM subsets (30). Thus, there may be a division of labor between these populations where airway TRM cells serve more of a sentinel function through the rapid production of cytokines to condition the local microenvironment and lung parenchymal TRM cells mediate direct killing of infected cells.
In addition to their cytolytic defect, it is intriguing that airway CD8 TRM cells fail to proliferate even when triggered by their cognate antigen. It was previously shown that airway CD8 TRM cells transferred intravenously into naïve hosts were capable of generating a complete secondary effector and memory response upon challenge, demonstrating that these are not terminally differentiated and are able to undergo clonal expansion (31). In contrast, our study examined proliferation in situ within the airways, where the local microenvironment does not provide abundant nutrient and growth factors to support an expanding T cell population. Clonal expansion of CD8 T cells following antigen stimulation is accompanied by a metabolic switch to glycolysis (32), and the concentration of glucose in airway fluid is 10–15 times lower than blood plasma (33, 34). Therefore, the inability of these cells to proliferate in the airways may simply be a consequence of insufficient nutrients within the local airway microenvironment.
Although IFN-γ-deficient mice show no defect in antiviral immunity following a primary influenza infection (28), its impact on protective cellular immunity to heterologous influenza challenge is less clear, with several conflicting reports regarding the protective role of IFN-γ during secondary challenge (35–38). Our data show that the inability of airway CD8 TRM cells to produce IFN-γ resulted in a significant increase in viral titers compared to wild-type airway CD8 TRM cells; it may be that the impact, positive or negative, of IFN-γ on protective immunity during influenza challenge depends on the timing of IFN-γ production. For example, it has been shown that IFN-γ production at the later stages of the acute response can lead to enhanced pathology (39), whereas our data suggest that early production of IFN-γ by airway CD8 TRM cells results in decreased levels of pro-inflammatory cytokines, likely due to decreased viral replication. It should also be noted that airway CD8 TRM cells also produced TNF-α and IL-10 and that these cytokines may account for the limited protective effect observed when IFN-γ-deficient airway CD8 TRM cells were transferred into the airways of naïve mice compared to PBS controls. In particular, the low levels of IL-10 produced may also limit early pro-inflammatory cytokine production and decrease pathology (40).
In summary, we show that airway CD8 TRM cells are sufficient to limit early viral replication following secondary influenza virus challenge, resulting in an attenuated duration of pro-inflammatory cytokine expression that can promote immunopathology. Furthermore, the protective efficacy was dependent on IFN-γ production by airway CD8 TRM cells and did not require local proliferation or enhanced cytolytic activity. We believe these data support the idea that antigen-specific airway CD8 TRM cells act as sentinels capable of rapidly responding to invading pathogens and alerting the immune system. Identifying approaches to generate or boost this airway CD8 TRM population through targeted vaccines and immunotherapies may afford greater protection against respiratory pathogens.
We would like to thank the Trudeau Institute Molecular Biology Core Facility and the NIH Tetramer Core Facility (contract HHSN272201300006C) for provision of MHC I tetramers and the Emory Children’s Pediatric Research Center Flow Cytometry Core for help with cell sorting. We would also like to thank Drs. David Woodland and Jeremy Boss for helpful discussions and critical review of the manuscript.
This work was supported in part by the Centers of Excellence in Influenza Research and Surveillance (CEIRS) contract number HHSN266200700006C (to J.E.K.), NIH HL122559 (to J.E.K.) and funds from Emory University. S.R.M. was supported in part by NIH F30HL118954 and NIH T32 AI007610-14.
The authors have no financial conflicts of interest.