Research ArticleINFECTIOUS DISEASE

Dynamics of influenza-induced lung-resident memory T cells underlie waning heterosubtypic immunity

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Science Immunology  06 Jan 2017:
Vol. 2, Issue 7, eaag2031
DOI: 10.1126/sciimmunol.aag2031

Getting the lung to remember flu

After influenza infection, some memory T cells enter the lung and become resident memory T cells (TRM). Although TRM play an important role in conferring protection against subsequent flu infections, TRM in the lung do not persist. Understanding how to improve TRM persistence is a longstanding goal in flu vaccination. Using a mouse model of flu, Slütter et al. report that TRM in the lung are prone to die and need to be constantly replenished from the circulating pool of memory T cells. Because circulating memory T cells wane with time, the lung eventually runs out of TRM. Given that immune cell infiltration can interfere with breathing, the lung may have evolved to limit immune cell residence.

Abstract

Lung-resident memory CD8 T cells (TRM) induced by influenza A virus (IAV) that are pivotal for providing subtype-transcending protection against IAV infection (heterosubtypic immunity) are not maintained long term, causing gradual loss of protection. The short-lived nature of lung TRM contrasts sharply with long-term maintenance of TRM induced by localized infections in the skin and in other tissues. We show that the decline in lung TRM is determined by an imbalance between apoptosis and lung recruitment and conversion to TRM of circulating memory cells. We show that circulating effector memory cells (TEM) rather than central memory cells (TCM) are the precursors for conversion to lung TRM. Time-dependent changes in expression of genes critical for lymphocyte trafficking and TRM differentiation, in concert with enrichment of TCM, diminish the capacity of circulating memory CD8 T cells to form TRM with time, explaining why IAV-induced TRM are not stably maintained. Systemic booster immunization, through increasing the number of circulating TEM, increases lung TRM, providing a potential new avenue to enhance IAV vaccines.

INTRODUCTION

Seasonal influenza vaccination can provide effective antibody-mediated protection when the main surface antigen [hemagglutinin (HA)] in the vaccine matches that year’s circulating influenza A virus (IAV) strains. However, mutations in the globular head region of HA can reduce the neutralizing capacity of vaccine-induced antibodies (1). In the absence of neutralizing antibodies, IAV-specific memory CD8 T cells, which are maintained systemically and in the lung (2), correlate with some degree of control of viral titers and reduction of disease symptoms in humans (3, 4). Mouse models suggest that it is the lung-resident memory CD8 T cells (TRM) that enable swift and robust protection against IAV infection (58). Thus, establishing a robust long-term TRM population should be an important goal for an IAV vaccine.

Nonetheless, major knowledge gaps remain concerning lung TRM generation, maintenance, and effector function, especially compared with TRM found in other nonlymphoid tissues such as skin, intestine, and female reproductive tract. Mouse studies suggest that lung TRM share common traits with TRM from other tissues, most notably the expression of the transmembrane C-type lectin CD69 and αE integrin CD103 (7, 9). However, at the transcriptome level, lung TRM are distinct from skin TRM or intestinal TRM (10), suggesting differential adaptation to specific microenvironments. One clear difference is that, compared with TRM populations in other tissues (11), lung TRM have a limited longevity, which strongly correlates with waning of subtype-transcending heterosubtypic immunity to IAV (7). Therefore, understanding the maintenance of lung TRM may provide information that can be used to extend the life span of this population, thus prolonging heterosubtypic immunity to IAV.

RESULTS

Limited longevity of IAV-induced lung TRM

Local infections, such as herpes simplex and vaccinia virus (Vac), have been used to study the formation and maintenance of TRM in the skin (12). Skin TRM have a distinct phenotype, coexpressing CD69 and CD103, molecules that have functional roles in retention in the tissues (10). Moreover, compared with non-TRM, skin TRM express low levels of a key transcription factor, Eomesodermin (Eomes) (13). The population of skin TRM is stably maintained (11) independently from the circulating memory CD8 T cell pool (12). In contrast, IAV-specific TRM are lost from the lung with time (7).

To address this discrepancy under conditions of a fixed T cell receptor (TCR) response, we transferred a small number of naive Thy1.1 P14 transgenic CD8 T cells [specific for the GP33 epitope of lymphocytic choriomeningitis virus (LCMV)] into naive Thy1.2 C57Bl/6 recipients followed by epicutaneous (EC) infection of the ear with Vac expressing the LCMV GP33 epitope (Vac-GP33) or intranasal (IN) infection with IAV (PR8-GP33). An intravascular (IV) stain with anti-CD45 antibody was applied before euthanasia to distinguish P14 cells in the tissue (IV) from those remaining in small capillary beds (IV+) (14, 15). In line with previous work (11), after an initial decline, the number of Vac-GP33–induced skin TRM (defined as CD69+CD103+ P14 cells) and the total number of IV P14 stabilized out to 200 days after infection (Fig. 1, A and B). We also confirmed that Vac-GP33–induced skin TRM display stable and low expression of Eomes (fig. S1, A and B).

Fig. 1 Reduced longevity of IAV-induced lung TRM compared with Vac-induced skin TRM.

C57Bl/6 mice (Thy1.2) were seeded with naive P14 transgenic T cells (Thy1.1) and infected with a sublethal dose of Vac-GP33 epicutaneously (EC) (A and B) or PR8-GP33 IN (C and D). At the indicated days after infection, mice were injected with fluorescently labeled anti-CD45.2 monoclonal antibody 3 min before euthanasia and tissue harvest. (A) Representative flow plots of P14 cells from the ear (skin) identified at various time points after EC infection with Vac-GP33 (top), IV stain (middle), and CD69 and CD103 expression on IV P14 (bottom). (B) Total number of IV P14 and CD69+CD103+ P14 cells in the ear parenchyma. n = 3 mice per time point. Representative of two independent experiments. Error bars represent means ± SEM. (C) Representative flow plots of P14 cells from the lung at various time points after PR8-GP33 infection (top), IV stain (middle), and CD69 and CD103 expression on IV P14 (bottom). (D) Total number of memory P14 and CD69+CD103+ P14 in the lung parenchyma. n = 3 to 4 mice per time point. Representative of three independent experiments. Error bars represent means ± SEM. (E) Representative flow plots of NP366 tetramer staining of lung cells at various time points after sublethal IAV PR8 infection (top), IV stain (middle), and CD69 and CD103 expression on IV NP366-specific CD8 T cells (bottom). (F) Total number of memory IV NP366+ and NP366+CD69+CD103+ CD8 T cells in the lung parenchyma. n = 3 mice per time point. Representative of two independent experiments. Error bars represent means ± SEM. (G) Representative CD69 and CD103 expression of IV NP366 tetramer–stained lung cells 30 and 125 days after IAV X31 infection. (H) C57Bl/6 mice were infected with H3N2 X31 IAV and, 30 or 125 days later, exposed to heterologous challenge with H1N1 PR8. D30 challenge was performed with and without CD8 T cell depletion. Lung PR8 virus titers measured in naive and X31-infected mice. n = 4 to 5 mice per group. Representative of two independent experiments. Error bars represent means ± SEM. **P < 0.01; ****P < 0.0001, multiple comparison one-way ANOVA.

However, lung TRM exhibited a different pattern. After an initial drop, there was an additional ~10-fold reduction in numbers of IV P14 cells from 50 to 200 days after infection (Fig. 1, C and D). In contrast to the skin, the number of P14 TRM in the lung parenchyma declined ~500-fold over the same time period (Fig. 1, C and D). IAV-induced lung TRM exhibited the same stable and low Eomes expression as Vac-induced skin TRM (fig. S1, C and D). Thus, despite their transience, IAV-generated lung IV CD69+CD103+ CD8 T cells represented bona fide TRM.

Waning of lung TRM was not limited to P14 TCR transgenic cells or recombinant IAV because endogenous TRM specific for an influenza nucleoprotein–derived epitope (NP366) also underwent ~500-fold decrease over the course of 200 days after IN PR8 infection (Fig. 1, E and F). The marked decline of IAV-induced lung TRM was not virus strain–dependent because NP366-specific lung TRM declined in a similar manner by day 125 after IN infection with X31 IAV (Fig. 1G). Moreover, the observation of limited lung TRM longevity extended to respiratory infections other than IAV. IN infection of P14 recipients with Vac-GP33 induced GP33-specific and endogenous Vac B8R–specific lung TRM responses, which declined in a similar fashion as IAV-induced lung TRM (fig. S2, A to D). Thus, the differences between lung and skin TRM are not explained by the use of different viruses for each organ.

Last, we confirmed that waning of IAV-induced lung TRM correlated with loss of CD8 T cell–mediated heterosubtypic protection. Primary exposure to H3N2 X31 IAV induced protection against challenge with heterosubtypic H1N1 PR8 IAV performed 30 days after X31 infection, measured by reduction of lung PR8 virus titers (Fig. 1H). Depletion of CD8 T cells rendered X31-immunized mice incapable of controlling PR8 virus titers. Heterosubtypic protection was completely lost at 125 days after initial X31 infection, which correlated with a substantial decline in lung TRM (Fig. 1, G and H).

In summary, these data showed that, in models of fixed TCR specificity with P14 TCR transgenic T cells, skin and lung TRM populations exhibit differences in persistence and confirmed the observation that IAV-specific TRM populations were not stable in the lung (7). In turn, the loss of TRM correlated with reduced CD8 T cell–dependent control of heterosubtypic IAV challenge (7), emphasizing the importance of understanding why the numbers of lung TRM decreased over time.

Apoptosis, rather than migration, drives the loss of IAV-generated TRM from the lung

To assess the possibility that some TRM migrate out of the lung and to determine the persistence of lung TRM, we labeled lung-residing cells using an in vivo carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling approach (16, 17). IN inoculation of CFSE into mice previously infected with PR8-GP33 labeled all P14 cells in the lung parenchyma but led to a negligible CFSE staining of P14 cells in the draining lymph nodes and the spleen (fig. S3), allowing for specific fate tracking of lung TRM. In vivo CFSE labeling of lung IV P14 generated by intraperitoneal (IP) infection with LCMV Armstrong, an approach that does not generate lung TRM (fig. S4), showed loss of labeled cells within 24 hours (Fig. 2, A and B). In contrast, IV P14 cells generated through IN PR8-GP33 infection persisted with a half-life of ~5 days (Fig. 2, A and B). Loss of CFSE did not result from proliferation, because no discernable 5-bromo-2′-deoxyuridine incorporation was detected in the CFSElo cells during this time period (fig. S5). To assess whether TRM are lost by migration, we blocked entrance of lung TRM to the afferent lymphatics using the S1PR1 agonist FTY720 (18). FTY720 treatment did not prevent the loss of CFSE+ P14 from the lung parenchyma (Fig. 2C), strongly suggesting that migration to the afferent lymph was not the primary mode of loss of IAV-generated TRM.

Fig. 2 Apoptosis, rather than migration, from the lung loss of CFSE-labeled IAV-induced lung TRM.

C57Bl/6 mice (Thy1.2) were seeded with naive P14 transgenic CD8 T cells (Thy1.1) and infected with PR8-GP33 IN (A to I), LCMV IP (A and B), or Vac-GP33 ID (D and F). Thirty days after infection, mice received IN CFSE and IV P14 cells were analyzed at the indicated time points (A to C). (A) Representative flow plots of CFSE labeling of IV P14 cells at selected time points and (B) cumulative analysis of the percentage of CFSE-labeled cells over time. n = 3 to 5 mice per time point. Representative of two independent experiments. Error bars represent means ± SEM. ***P < 0.001, unpaired t test. (C) After IN CFSE labeling, mice received daily injection of FTY720 or vehicle control. Cumulative percentage of IV CFSE+ P14 at selected time points after CFSE staining. n = 3 mice per time point. Representative of two independent experiments. Error bars are means ± SEM. (D) Flica (caspase 3/7) staining of IV+ lung/blood (red) and IV lung/skin (black) P14 cells 30 days after IN PR8-GP33 or EC Vac-GP33 infection. Left, representative profiles; right, cumulative data. n = 4 mice per group. Representative of two independent experiments. Error bars are means ± SEM. P = 0.0003, multiple comparison one-way ANOVA. (E) Representative plots (left) and cumulative results (right) of Flica (caspase 3/7) staining in different subsets of D30 IV memory P14 cells isolated from the lungs of IAV PR8-GP33–infected mice. n = 4 mice per group. Representative of two independent experiments. Error bars represent means ± SEM. P = 0.001, multiple comparison one-way ANOVA. (F) Bcl-2 expression assessed 30 days after infection with PR8-GP33 or Vac-GP33 in IV+/blood (red) and IV (black) P14 cells from the lung or skin. Left, representative plots; right, cumulative data. n = 4 mice per group. Representative of two independent experiments. Error bars represent means ± SEM. **P = 0.004, unpaired t test. GMFI, geometric mean fluorescence intensity. (G) Representative plots (left) and cumulative results (right) of Bcl-2 staining in different subsets of D30 IV memory P14 cells isolated from the lungs of IAV PR8-GP33–infected mice. Reference is Bcl-2 expression by IV+ P14 cells in the lung (red). n = 4 mice per group. Representative of two independent experiments. Error bars represent means ± SEM. P < 0.0001, multiple comparison one-way ANOVA. (H) Decrease in mitochondrial membrane potential (ΔΨ mito) assessed 30 days after infection with PR8-GP33 by Mito Flow staining in IV+ (red) and IV (black) P14 cells from the lung. Left, representative plots; right, cumulative data. n = 3 mice per group. Representative of two independent experiments. Error bars represent means ± SEM. ***P = 0.0002, unpaired t test. (I) Representative plots (left) and cumulative results (right) of Mito Flow staining in different subsets of D30 IV memory P14 cells isolated from the lungs of IAV PR8-GP33–infected mice. n = 3 mice per group. Representative of three independent experiments. Error bars represent means ± SEM. P < 0.0001, multiple comparison one-way ANOVA.

Alternatively, impaired maintenance of lung TRM could result from an increased apoptosis. In support of this notion, we observed a higher percentage of IAV-induced IV lung memory P14 expressing active caspases 3/7 (Flica stain) relative to IV+ lung memory P14, suggesting elevated proapoptotic activity in tissue-embedded cells (Fig. 2D). Increased apoptosis is not a general feature of TRM, because we observed no difference in Flica staining between skin IV memory P14 cells and their IV+ (blood) counterparts (Fig. 2D). Furthermore, lung IV CD103+ P14 cells displayed higher caspase 3/7 activity than did CD103 P14 cells (Fig. 2E). In addition, protein expression of the antiapoptotic molecule Bcl-2 was substantially reduced in IV memory P14 cells relative to the IV+ cells in the lung or in blood (Fig. 2F). Decreased Bcl-2 protein expression in IV relative to IV+ memory P14 cells was also observed at day 90 after infection, suggesting that compromised survival may be a sustained characteristic of lung TRM (fig. S6). In contrast, skin IV and IV+ circulating memory P14 cells exhibited similar levels of Bcl-2 expression at day 30 after infection (Fig. 2F). Notably, within the lung IV P14 population, CD103+ cells had substantially decreased Bcl-2 expression compared with IV+ P14, whereas CD103 P14 cells had similar Bcl-2 expression to IV+ cells (Fig. 2G). Last, we assessed the maintenance of mitochondrial membrane potential (ΔΨ mito) to evaluate the proapoptotic state of memory CD8 T cells in the lung after IAV infection (Fig. 2, H and I). In line with increased Flica staining and decreased Bcl-2 expression, a larger fraction of lung IV P14 cells exhibited decreased ΔΨ mito fluorescence compared with IV+ P14 (Fig. 2H). In addition, IV CD103+ cells showed lower ΔΨ mito fluorescence compared with their IV CD103 counterparts (Fig. 2I). Together, these data showed that lung TRM were prone to apoptosis, suggesting that cell death, rather than migration to draining lymph nodes, contributed to the limited longevity of this CD8 T cell population.

Lung TRM maintenance is linked to persistence of circulating memory CD8 T cell pool

Despite the increased signature of apoptosis in the TRM population (Fig. 2, D to I) and a steady decline of CFSE-labeled TRM P14 cells in the lung parenchyma (Fig. 2B), the total number of TRM did not decrease over the 6-day CFSE chase interval (fig. S7). As depicted in Fig. 3A, the emergence of CFSE, CD69+, or CD103+ P14 over time compensated for the loss of CFSE-labeled memory P14 from the lung TRM pool. Because proliferation was eliminated as a cause of disappearance of CFSE+ memory P14 cells (fig. S5), these data suggest de novo generation of lung TRM from a memory population outside of the lung parenchyma. Given previous reports showing continuous recruitment of circulating memory CD8 T cells into the lung even weeks after the clearance of the infection (19, 20), we hypothesized that lung TRM were continuously formed from circulating precursors. Systemic antibody-mediated depletion has been routinely used to show that, in most tissues, TRM are maintained independently from the circulating memory population (21, 22). However, antibody depletion of the systemic memory CD8+ T cell population also depleted lung TRM (fig. S8). As an alternative, we used a system where depletion is based on cellular interactions (21). Male and female P14 cells were transferred in a 1:1 ratio into female recipients, which were subsequently infected either IN with PR8-GP33 or EC with Vac-GP33 (Fig. 3B). Male P14 cells underwent initial expansion in numbers like female P14 cells but were rejected from the circulation after 2 weeks (Fig. 3C). As previously reported (21), male P14 cells in the skin were detectable for several weeks after they were systemically rejected (Fig. 3, D and E), indicating maintenance of skin TRM without input from circulating memory CD8 T cells. Male P14 cells were also detectable in the lung parenchyma for several weeks after systemic rejection (Fig. 3, D and E), suggesting that the parenchyma provides a niche that shields male P14 from deletion. However, the 1:1 ratio is not maintained long term, and male P14 cells are progressively lost from the lung parenchyma (Fig. 3, D and E). Although we cannot formally exclude the possibility that anti-male cytotoxic T lymphocytes gradually killed male P14 cells, in conjunction with the observed apoptosis of lung TRM (Fig. 2, D to I), these data strongly suggested that lung TRM were not adequately maintained without input from the circulating memory CD8 T cell population and that maintenance of the lung TRM pool required continuous recruitment of circulation-derived precursors. Consistent with this hypothesis, blocking the entrance of circulating memory P14 into the lung parenchyma by administration of pertussis toxin (PTx) 3 weeks after infection significantly (P < 0.05) reduced the number of TRM in the lung parenchyma 1 week later. In contrast, PTx treatment did not affect the number of systemic memory P14 in the spleen (Fig. 3F). These data are consistent with the requirement for a continuous influx of memory P14 cells from the circulation to maintain stable lung TRM numbers over the short-term period.

Fig. 3 Maintenance of IAV-induced lung TRM depends on circulating memory CD8 T cells.

(A) Representative example of CD69 (top) and CD103 (bottom) expression on CFSE+ and CFSE IV P14 cells in the lung at the indicated time points after IN CFSE labeling. (B) Female mice were seeded with male and female naive P14 cells in a 1:1 mixture and were infected IN with PR8-GP33 or EC with Vac-GP33. (C) Percentage of female and male P14 cells in blood after infection with IAV PR8-GP33. n = 3 to 7 mice per group. Representative of three independent experiments. Error bars represent means ± SEM. (D) Representative flow plots depicting the proportion of female (Thy1.1/1.1) and male (Thy1.1/1.2) P14 cells in spleen, lung, or skin at various days after Vac-GP33 (left) or PR8-GP33 (right) infection. (E) Cumulative data depicting ratio of male/female P14 in indicated organs at indicated times after infection. n = 3 to 4 mice per group. Representative of three independent experiments. Error bars represent means ± SEM. One-way ANOVA: *P < 0.05, **P < 0.01, ****P < 0.0001 compared with spleen; ##P < 0.01, ####P < 0.0001 compared with lung. (F) C57Bl/6 mice were seeded with congenically labeled naive P14 (Thy1.1) cells and infected with a nonlethal dose of X31-GP33. After 3 weeks, mice received 1 μg of PTx every other day for 1 week. Number of CD69+CD103+ P14 in the lung parenchyma and total number of P14 in the spleen after PBS or PTx treatment. n = 3 mice per group. Representative of two independent experiments. Error bars represent means ± SEM. ***P = 0.0009, unpaired t test. NS, not significant.

Continuous seeding of lung TRM by precursors from the circulation is influenced by the lung cytokine milieu

Continuous formation of lung TRM has been proposed (23, 24) and was thought to be driven by persisting IAV antigen. Antigen has been shown to persist for at least 2 months in the lung after IAV infection (24), making this an attractive explanation for the continuous formation but also the eventual disappearance of lung TRM (7). To confirm continuous recruitment of circulating memory CD8 T cells to the lung and to assess whether persisting antigen drives conversion of recruited memory CD8 T cells to lung TRM, we adoptively transferred spleen-derived (CD69CD103) memory P14 cells that were generated by X31-GP33 infection into naive, PR8 (no antigen)–infected, or PR8-GP33 (antigen)–infected mice (Fig. 4A). Analysis of the lung 1 week after transfer revealed de novo generation of TRM in all groups of mice (Fig. 4B). A substantially larger fraction of transferred P14 cells entered the lung parenchyma of PR8- and PR8-GP33–infected mice compared with naive mice (Fig. 4C). This suggested that the lung environment still drives additional recruitment of P14 cells into the parenchyma weeks after IAV infection, but this process does not require cognate antigen. In addition, conversion to a TRM phenotype was higher in PR8- and PR8-GP33–infected mice compared with naive mice, suggesting that the post-IAV lung environment was permissible to de novo TRM generation weeks after infection (Fig. 4D). However, no difference in P14 conversion to TRM was observed between PR8- and PR8-GP33–infected mice. Thus, the IAV-experienced lung contains cues other than antigen to drive formation of lung TRM from circulating precursors. Cytokines such as transforming growth factor–β, interleukin-33 (IL-33), and tumor necrosis factor (TNF) have been implicated in formation of TRM (10, 25), with IL-33 specially implicated in the function of lung-resident innate lymphocyte cells (26). Because IAV infection increased lung IL-33 (27) and TNF (28) (fig. S9), we transferred memory P14 CD8 T cells derived from spleens of PR8-GP33–infected mice into naive recipient mice (Fig. 4E) that were treated or not with blocking (anti–IL-33R) or neutralizing (anti-TNF) antibodies before analyses on day 7 (Fig. 4E). Blocking the IL-33 receptor reduced the number IV P14 in the lung parenchyma (Fig. 4, F and G) but did not affect conversion to a TRM phenotype (Fig. 4, F and H). On the other hand, neutralization of TNF reduced both the accumulation (Fig. 4, F and G) of P14 cells in the lung parenchyma and acquisition of TRM phenotype (Fig. 4, F and H), suggesting a role for this cytokine in formation and maintenance of lung TRM.

Fig. 4 Circulating memory CD8 T cells are recruited and converted to TRM in the IAV-experienced lung in an antigen-independent fashion.

(A) Memory P14 (Thy1.1) cells were isolated from spleens of X31-GP33–infected mice and transferred into naive mice or mice previously infected with PR8 or PR8-GP33 (Thy1.2). (B) Representative flow plots of P14 staining (top), IV staining (middle), and CD69 and CD103 staining in IV P14 population (bottom) in the lung. Cumulative percentage of IV P14 (C) and percentage of CD69+CD103+ P14 (D) in the lung parenchyma. n = 6 mice per group. Representative of two independent experiments. Error bars represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, multiple comparison one-way ANOVA. (E) Memory P14 (Thy1.1) cells were transferred into mice previously infected with PR8. Mice were IN treated with 100 μg of rat IgG, anti-ST2, or anti-TNF on days 0, 2, and 4 after transfer. (F) Representative flow plots of P14 staining (top), IV staining (middle), and CD69 and CD103 staining in IV populations (bottom) in the lung. Cumulative percentage of IV P14 (G) and percentage of IV CD69+CD103+ P14 (H) in the lung. n = 8 to 9 mice per group. Cumulative results from two independent experiments. Error bars represent means ± SEM. *P < 0.05, **P < 0.01, multiple comparison one-way ANOVA.

Thus, our data show that the maintenance of IAV-induced lung TRM CD8 T cell population critically depended on the circulatory CD8 T cell memory pool. Continuous seeding of the lung TRM niche by circulating memory CD8 T cells appeared to be antigen-independent and at least partially driven by local inflammatory cues (e.g., IL-33 and TNF).

IAV-induced circulating memory CD8 T cells lose the capacity to form TRM over time

The capacity of circulating memory CD8 T cells to replenish lung TRM contradicts the observation that TRM are lost by about 6 months after infection. Therefore, we hypothesized that, with time, circulating IAV-specific memory CD8 T cells lose their intrinsic capacity to generate TRM. To probe this, we transferred memory P14 cells isolated from spleens of mice infected with PR8-GP33 for 20 to 30 days (early memory) or >100 days (late memory) into recipient mice infected with PR8 virus 21 days before (Fig. 5A). Late memory P14 cells showed a marked decrease in accumulation in the lung parenchyma relative to early memory P14 (Fig. 5B). Furthermore, late memory P14 displayed poor conversion to a TRM phenotype compared with early memory P14 (Fig. 5C). These data suggest that circulating memory CD8 T cells intrinsically lose the capacity to form lung TRM over time, independent of the change in the local inflammatory cues.

Fig. 5 IAV-induced circulating memory CD8 T cells lose the capacity to form lung TRM with time.

(A) Memory P14 (Thy1.1) cells were isolated from spleens of mice infected with PR8-GP33 20 days (early memory) or >100 days before (late memory). Isolated cells were transferred into mice (Thy1.2) previously infected with PR8. (B) Representative flow plots (top) and cumulative data (bottom) of IV staining of P14 cells from the lung 7 days after transfer. (C) Representative flow plots (top) and cumulative data (bottom) of CD69 and CD103 staining of IV P14 cells in the lung. n = 3 to 5 mice per group. Representative of two independent experiments. Error bars represent means ± SEM. *P < 0.05 **P < 0.01, unpaired t test.

To assess the molecular signatures that could potentially explain the difference between early and late circulating memory CD8 T cells in forming lung TRM, we analyzed genome-wide mRNA expression in early (20 to 30 days after infection) and late (>100 days after infection) spleen-derived memory P14 generated by IN PR8-GP33 infection. Many genes (2657; based on ≥1.25-fold difference, P < 0.05) were differentially expressed in late versus early memory P14, with a similar number being up-regulated (1326) or down-regulated (1331) (fig. S10A). Notably, transcriptome analysis suggested differential expression of three transcription factors identified as master regulators of TRM formation: mRNA for Eomes (1.68× up-regulated in late versus early memory P14), Blimp-1 (2.19× down-regulated in late versus early memory P14), and Hobit (1.21× down-regulated in late versus early memory P14) (13, 29). Real-time quantitative polymerase chain reaction (qPCR) analysis performed on mRNA isolated from early and late IAV-induced memory P14 demonstrated increased Eomes (P < 0.0001) and decreased Blimp-1 (P = 0.021) and Hobit (P = 0.0033) mRNA expression in late versus early splenic memory P14 cells (Fig. 6A). Intracellular protein staining confirmed that a substantially higher percentage of late compared with early memory P14 cells up-regulated Eomes (Fig. 6B). Combined, these data show that the transcription factor profile of late memory P14 cells was incompatible with acquisition of TRM phenotype.

Fig. 6 Late IAV-induced memory CD8 T cells display transcription factor profile unfavorable for TRM formation.

(A) Relative mRNA expression of Eomes, Prdm-1 (Blimp-1), and Hobit by qPCR using RNA isolated from spleen-derived memory P14 cells 20 days (early memory) or >100 days (late memory) after IN PR8-GP33 infection. n = 5 RNA sample pools per group. Representative of two independent experiments. Error bars represent means ± SEM. *P = 0.021, **P = 0.003, ****P < 0.0001, unpaired t test. AU, arbitrary units. (B) Eomes intracellular flow cytometry staining of early and late spleen-derived memory P14 cells (white) compared with naive P14 cells (gray).

In addition, ingenuity pathway analyses revealed substantial alterations in a leukocyte migration gene set between late and early memory P14, represented by 158 genes (P = 5.48 × 10−25) (table S1). Gene set enrichment analysis showed that leukocyte migration–associated genes were significantly negatively enriched (false discovery rate < 0.01; normalized enrichment score, −1.45) in late memory P14 (fig. S10B), suggesting some compromise in the ability to migrate to tissues. To further refine our gene analysis and focus only on T lymphocyte migration, we performed a literature-based selection of genes from the leukocyte migration pathways based on (i) expression by T lymphocytes and (ii) association with cell movement, migration, or chemotaxis. This approach shortlisted 70 genes that were further divided into specific categories, based on the function of their protein products (table S2). Notably, expression of most of these genes (>75%) was down-regulated in late versus early memory P14 (fig. S10C). Combined, the gene expression signatures strongly suggested that, with time, memory CD8 T cells down-regulate the complex functional network of molecules collectively controlling cell migration to peripheral tissues. In turn, the reduced capacity of late memory CD8 T cells to enter lung tissue likely contributed to the decline in the TRM population.

Our data showed that, with time, IAV-induced circulating memory CD8 T cells lose the capacity to form de novo lung TRM. We propose that inefficient conversion to TRM, based on an unfavorable transcription factor landscape, together with the decreased recruitment to the lung tissue due to loss of migratory capacity in late circulating memory CD8 T cells, underlies the waning of lung TRM.

Circulating effector memory CD8 T cells are precursors of IAV-induced lung TRM

Analyses of the leukocyte migration genes revealed that two of the relatively few mRNAs enriched in late memory P14 cells were those coding for CCR7 and CD62L (fig. S10C), well known to be required for homing to secondary lymphoid tissue and as canonical markers of central memory CD8 T cells (TCM) (30). In addition, we observed that the vast majority (>85%) of PR8-GP33–induced circulating memory P14 cells up-regulated CD62L by day 90 after infection. However, the TRM population in the lung was composed of predominantly CD62L cells (Fig. 7A). Because TRM P14 had a surface phenotype like effector memory T cells (TEM) (CD62L), it was very likely that they were recruited from the circulating TEM pool, although active down-regulation of CD62L upon recruitment to the lung could not be ruled out. To discriminate between these possibilities, we isolated PR8-GP33–induced splenic memory Thy1.1 P14 cells on day 65 after infection (when an optimal ratio of CD62L+ and CD62L P14 was observed), separated them into enriched CD62L+ and CD62L subpopulations, transferred them into mice infected IN with PR8 IAV 21 days earlier (Fig. 7B), and assessed their capacity to convert into TRM 7 days later. Notably, the TRM master regulator Eomes was differentially expressed in the donor populations, with CD62L+ P14 (blue) being Eomeshi and CD62L (red) expressing less Eomes (Fig. 7C). The vast majority (89%) of P14 cells isolated from the lungs of CD62L+ recipients were CD62L+, whereas most (76%) of the P14 cells in the lungs of CD62L recipients were CD62L (Fig. 7D). This finding strongly suggested that down-regulation of CD62L after cell entry into the lung was unlikely to account for the observed enrichment of TEM-like cells (CD62L) in the lung. Although P14 cells recovered from the lungs of CD62L recipients (red) were found in the blood (IV+) and parenchyma (IV), almost all P14 cells in the lungs of CD62L+ recipients (blue) were IV+, with only ~1% of cells residing in the parenchyma (Fig. 7E). A discernable fraction of IV P14 (~10%) formed CD69+CD103+ lung TRM in the CD62L recipients (Fig. 7F). In contrast, we did not detect any lung TRM P14 in CD62L+ recipients (Fig. 7F).

Fig. 7 Lung TRM are recruited from circulating TEM.

(A) CD62L expression by memory P14 cells (day 90) isolated from peripheral blood or by IV CD103+CD69+ P14 isolated from the lung. (B) Memory P14 (Thy1.1) cells were isolated from spleens of mice infected with PR8-GP33 65 days earlier. Cells were separated into CD62L+ and CD62L subpopulations as described, and 3 × 106 separated P14 cells were transferred into Thy1.2 recipients infected with PR8 21 days earlier. Recipient mice were sacrificed 7 days after the transfer, and lung P14 TRM were evaluated. (C) Eomes expression by CD62L+ (blue) and CD62L (red) P14 cells measured before separation. (D) CD62L expression by P14 cells isolated from the lungs of CD62L+ recipients (blue) and CD62L recipients (red). (E) Representative plots (left) and cumulative results (right) of IV staining of P14 cells isolated from the lungs of CD62L (red) and CD62L+ (blue) recipients. (F) Representative plots (left) and cumulative results (right) of CD69 and CD103 staining of IV P14 cells isolated from the lungs of CD62L (red) and CD62L+ (blue) recipients. n = 4 mice per group. Representative of two independent experiments. Error bars represent means ± SEM. **P = 0.0086, ***P = 0.0008, unpaired t test.

Thus, our results identified circulating TEM as the precursors for de novo formation of IAV-specific lung TRM. These data suggested the intriguing hypothesis that expansion of the circulating TEM population induced by IAV infection could increase numbers of lung TRM. To test this hypothesis, we exposed PR8-GP33 immune or naive P14 recipient mice to systemic infection with recombinant Listeria monocytogenes expressing GP33 (LM-GP33) or an unrelated epitope derived from the Plasmodium berghei TRAP protein (LM-TRAP) (31, 32) 45 days after the initial PR8-GP33 infection (Fig. 8A). Mice were sacrificed 30 days after the systemic boost (75 days after initial PR8-GP33 infection), and circulating and lung P14 cells were analyzed (Fig. 8A). As predicted, systemic boosting with LM-GP33 enhanced the fraction of circulating TEM P14 cells (~84% were CD62L), in sharp contrast to all the other immunization groups where circulating TCM P14 dominated the response (Fig. 8, B and C). As depicted in Fig. 8D, the most marked result of systemic boosting with LM-GP33 was an ~120× increase in the frequency of lung P14 memory cells, relative to nonboosted or LM-TRAP–infected controls. Notably, naive mice that received the systemic LM-GP33 boost generated a circulating memory P14 population but did not generate a detectable population of lung P14 TRM (Fig. 8, D and E). In addition, boosting IAV immune mice with LM-TRAP did not alter lung P14 TRM numbers. However, in support of our hypothesis, the large increase in circulating TEM in PR8-GP33 immune mice boosted with LM-GP33 was associated with an ~10-fold increase in lung P14 TRM at 30 days after boost (Fig. 8, D and E). The increase in lung TRM did not result from a marked increase in frequencies of CD69+CD103+ P14 cells in the lung IV population but rather from an increase in the total number of lung IV P14 cells in the LM-GP33–boosted mice (Fig. 8, D and E). Thus, systemic boosting of IAV-induced memory CD8 T cell responses substantially increased the frequency of circulating TEM, which in turn enhanced the seeding of IAV-experienced memory CD8 T cells in the lung. The sustained ability of the lung environment to convert TEM to TRM, in the context of a larger number of lung parenchymal TEM, resulted in a substantial increase in the number of lung TRM.

Fig. 8 Systemic vaccination boosts IAV-induced TRM pool by expanding circulating TEM.

(A) C57Bl/6 mice were seeded with naive P14 cells (Thy1.1) and IN infected with IAV PR8-GP33. Some mice received IV booster immunization with recombinant L. monocytogenes expressing GP33 (LM-GP33) or mock booster immunization with L. monocytogenes expressing a P. berghei–derived TRAP epitope (LM-TRAP) 45 days after initial IAV infection. At the same time, a separate group of C57Bl/6 mice was seeded with naive P14 CD8 T cells and IV infected with LM-GP33. Analysis was performed 30 days after the LM infections. Representative plots (B) and cumulative results (C) of CD62L expression measured in splenic P14 cells of mice from all four immunization groups. n = 4 mice per group. Representative of two independent experiments. Error bars represent means ± SEM. P < 0.0001, multiple comparison one-way ANOVA. (D) Representative flow plots of P14 cells (top), IV staining (middle), and CD69 and CD103 staining of IV P14 cells (bottom) in the lung. (E) Cumulative numbers of CD69+CD103+ P14 in the lung parenchyma. n = 4 mice per group. Representative of two independent experiments. Error bars represent means ± SEM. P = 0.005, multiple comparison one-way ANOVA. b.l.d., below limit of detection.

DISCUSSION

Here, we show that maintenance of lung TRM, in contrast to well-studied TRM populations in skin, is driven by a dynamic equilibrium between apoptosis in the lung parenchyma and reseeding from the circulatory TEM population. However, the capacity of the circulating memory CD8 T cell population to home to the lung and convert to TRM is lost with time, leading to the progressive loss of the lung TRM population and heterosubtypic protection. Of relevance for vaccine design, we show that increasing circulating TEM through systemic boosting can increase the size of the lung TRM pool, potentially prolonging heterosubtypic immunity.

Our data suggest that lung TRM are continuously being replenished from CD69CD103 circulating memory CD8 T cells in an antigen-independent and inflammation-dependent manner. These results are in concordance with earlier reports using parabiotic mice (24, 33) or adoptive transfers (5, 6), which show that memory CD8 T cells can migrate into the lung during the steady state and in the absence of antigen. However, these earlier studies did not use an IV stain to distinguish CD8 T cells in the lung parenchyma from those in the lung vasculature and did not use the markers to distinguish whether the lung CD8 T cells were transient effector memory subsets or became genuine TRM. The main findings of this study stem from comparison of TRM in the skin and the lung, induced by recombinant Vac-GP33 and PR8-GP33, respectively. Although this system allowed us to compare the responses of the same TCR transgenic T cells to the same antigen, the antigen was delivered by viruses with distinct biological interactions with the host. While acknowledging this complexity, we also show that TRM, specific for the same GP33 epitope or the endogenous Vac B8R epitope, also wane with time after respiratory Vac-GP33 infection, suggesting that the transient nature of lung TRM is mainly driven by the local tissue environment. Whether this finding will extend to all respiratory virus infections remains to be determined. Experimental support for the notion that circulating TEM can home to the IAV-conditioned lung and convert to lung TRM was obtained from multiple approaches, including pulse chase of lung TRM with lung CFSE labeling and the progressive loss of male P14 TRM in IAV-GP33–infected female mice, in contrast to their skin TRM counterparts. In these studies, we cannot formally rule out that some loss of CFSE-labeled lung TRM could occur through migration into the airways or that loss of male TRM in the lung could result from rejection by male-specific T cells reaching the lung parenchyma of the female mice. However, we also found that PTx treatment, which prevents migration of circulating memory CD8 T cells into tissues, also reduced lung TRM and that CD69CD103 splenic TEM homed to the lung and converted to TRM after adoptive transfer. Thus, the preponderance of evidence is consistent with the notion that the longevity of lung TRM after IAV infection involves replenishment from the pool of circulating memory CD8 T cells.

In addition to apoptosis of lung TRM and their replenishment from the circulating memory populations, we also show that circulating memory CD8 T cells lose the capacity to form lung TRM with time. Our data support a model where decreasing cellular input, in combination with the loss of existing populations through apoptosis, underlies the relatively slow decline of TRM in the lungs. We propose that, with time, circulating memory CD8 T cells acquire a transcription factor profile that may not be permissible for conversion to TRM in the lung (up-regulated Eomes and down-regulated Blimp-1 and Hobit) (13, 29). In addition, bioinformatic analyses emphasize a loss in general tissue migratory capacity in memory CD8 T cell populations with time, which supports our observation that circulatory memory CD8 T cells eventually lose the capacity to populate lung parenchyma.

Last, we identify the circulating TEM pool as the main source of precursors for lung TRM. These results suggest that the progressive shift of systemic memory CD8 T cells toward TCM, and concurrent loss of TEM populations, underlies the slow decay of IAV-induced lung TRM, although time-dependent changes in the TEM subset may also contribute to loss of TRM. Consistent with either notion, we show that systemic booster vaccination, through expansion of the circulating TEM population, can increase the numbers of lung TRM CD8 T cells. Despite the >100× increase in frequency of lung CD8 T cells, numbers of TRM increased only ~10-fold, suggesting that optimization of the booster approach may be possible.

In conclusion, we identify several key features of IAV-induced lung TRM maintenance that differentiate them from maintenance of TRM populations in other microenvironments. In addition, we provide an explanation for the gradual decline of lung TRM and loss of heterosubtypic protection with time, based on loss of these cells by apoptosis and the diminishing capacity of circulating memory CD8 T cells to home to the lung and convert to TRM. Further, we also show that waning lung TRM can be at least partially restored by systemic boosting to increase the size of the circulating TEM populations, suggesting that longevity of lung TRM may be manipulated through rational vaccination.

MATERIALS AND METHODS

Study design

The main aim of the study was to explain the gradual waning of IAV-induced lung TRM, which correlates with loss of heterosubtypic protection. For this purpose, we adopted a mouse model of IAV infection. The initial phase of the study confirmed the appropriateness of our model and developed approaches to evaluate the biological status and longevity of IAV-induced lung TRM. Subsequent studies addressed the biology underlying recruitment and conversion of circulating memory CD8 T cells into lung TRM, the waning of this ability with time, and the capacity of systemic booster immunization to restore lung TRM. All experiments were performed at least twice. The study involved sublethal infections with IAV, Vac, or euthanasia before excessive body weight loss after IAV challenge; thus, no predetermined outcomes, such as weight loss, were used in the study, and no outliers were excluded from the data analyses.

Mice

C57Bl/6 mice were originally derived from the National Cancer Institute (Fredericksburg, MD), and a colony is maintained in house. P14 transgenic mice (on a C57Bl/6 background) were acquired from the Jackson Laboratory. All animal studies and procedures were approved by the University of Iowa Animal Care and Use Committee, under U.S. Public Health Service assurance, Office of Laboratory Animal Welfare guidelines.

Viral and bacterial infections

Influenza A/PR/08/34 H1N1 (PR8) and recombinant PR8 or X31 expressing GP33 (34, 35) were grown in chicken eggs. Allantoic fluid was diluted in phosphate-buffered saline (PBS), and mice received a sublethal dose [2 × 104 TCID50 (median tissue culture infectious dose)] while lightly anesthetized. EC infection with Vac expressing GP33 (Vac-GP33, a gift by E. J. Wherry, University of Pennsylvania) was performed by applying 5 × 106 plaque-forming units (PFU) of the virus on the center of the ear pinna, followed by poking 25 times with 27-gauge needle. For IN infection with Vac-GP33, mice were inoculated with 107 PFU of the virus. LCMV Armstrong infections were performed by IP injection of 2 × 105 PFU of the virus. Systemic booster/mock booster immunizations were performed by IV injection of 107 colony-forming units of recombinant attenuated L. monocytogenes expressing GP33 (LM-GP33) or a P. berghei TRAP-derived epitope (LM-TRAP) (31).

Influenza protection and lung virus titers

C57Bl/6 mice were infected with X31 IAV (a reassortant IAV with six internal genes of PR8 and the HA and neuraminidase of H3N2 A/Aichi/2/68). Heterosubtypic protection by early memory CD8 T cells was assessed 30 days after infection. Half of the mice were inoculated with CD8 depleting antibody, clone 2.43 (400 μg IP and 100 μg IN), whereas the rest of the animals were treated with equal amounts of rat immunoglobulin G (IgG) control antibody. Two days after depletion, X31-immune mice and naive control animals were IN challenged with heterosubtypic PR8 (H1N1) virus. Lungs were harvested, and virus titers were assessed 3 days after challenge. Protection by late memory CD8 T cells was assessed in the same way, with PR8 challenge performed >100 days after the initial X31 infection.

Intravascular staining and tissue preparation

Mice were intravenously injected with 2 μg of anti-CD45.2–allophycocyanin (clone 104, BioLegend) in PBS. After 3 min, mice were euthanized, and whole lung or skin (ear) was isolated. Lung and skin were cut into small pieces and incubated for 1 hour in a mixture of collagenase (125 U/ml) and deoxyribonuclease (0.1 mg/ml) at 37°C. Single-cell suspensions were obtained by forcing the organs through a 70-μm mesh screen. Erythrocytes were lysed using Vitalyze (BioE), and leukocytes were purified with 35% Percoll (GE Healthcare) in Hanks’ balanced salt solution.

Statistical analysis

Comparison between two study groups was statistically evaluated by unpaired, two-tailed t test. Comparisons between more than two groups (single factor) were evaluated using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison post hoc test. Two-way ANOVA with Sidak’s multiple comparison post hoc test was used to evaluate comparison between more than two groups at different time points (multiple factors). In all the tests performed, statistical significance was quantified as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Statistical analyses were performed using GraphPad Prism 7 software. Additional statistical information including P values is listed in table S3.

SUPPLEMENTARY MATERIALS

immunology.sciencemag.org/cgi/content/full/2/7/eaag2031/DC1

Materials and Methods

Fig. S1. IAV-induced lung TRM express low levels of Eomes.

Fig. S2. Vac-induced lung TRM are transient.

Fig. S3. In vivo CFSE labeling of the lung.

Fig. S4. LCMV Armstrong infection does not induce lung TRM.

Fig. S5. CFSE dilution in the lung is not a consequence of active cell proliferation.

Fig. S6. Bcl-2 expression in late lung memory P14 cells.

Fig. S7. Total numbers of CD69+ and CD103+ cells in the lung parenchyma do not change up to 6 days after IN CFSE labeling.

Fig. S8. Deletion of lung TRM CD8 T cells after systemic administration of anti-CD8 antibody.

Fig. S9. Levels of IL-33 and TNF in lungs at different stages after IAV infection.

Fig. S10. Early versus late IAV-induced memory CD8 T cells differentially express genes involved in lymphocyte migration.

Table S1. Gene set of leukocyte migration pathway differentially expressed in late versus early memory P14 cells.

Table S2. Selected list of genes from the leukocyte migration pathway.

Table S3. Raw data sets and statistical analyses.

REFERENCES AND NOTES

Acknowledgments: We thank members of the Harty laboratory for insightful discussion and E. J. Wherry (University of Pennsylvania) for the recombinant Vac. Funding: This work was supported by NIH grants AI 42767 and AI 114543 to J.T.H. Author contributions: B.S., N.V.B.-B., G.A., S.S.-A., and J.T.H. designed experiments. B.S. and N.V.B.-B. performed experiments and data analysis. G.A. performed IN Vac infection experiments and data analysis. S.M.V. provided crucial experimental reagents. B.S., N.V.B.-B., G.A., S.S.-A., S.M.V., and J.T.H. contributed to writing and editing of the manuscript. B.S. and N.V.B.-B. performed statistical analysis. Competing interests: The authors declare that they have no competing interests. Data and materials availability: Microarray data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus database under the accession number GSE86973.
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