Cutting Edge: Tissue-Retentive Lung Memory CD4 T Cells Mediate Optimal Protection to Respiratory Virus Infection

Migration of activated T cells to peripheral tissue sites during infection promotes local immune responses to clear pathogens. After clearance, long-lived, pathogen-specific memory T cells can take up residence and recirculate within multiple lymphoid and nonlymphoid compartments, with significant populations in mucosal sites such as lung and intestine (1, 2). The presence of memory T cells in tissue sites has been associated with increased protection to virus infections in mice (1–3); however, it has been difficult to establish whether in situ immunity is due to T cells resident in the tissue versus those rapidly recruited from other sites. Defining the protective T cell subsets for specific compartments is critical to the successful design of vaccines that target tissue sites.

Respiratory infection with influenza virus results in the generation and dissemination of memory T cells into lung and lymphoid tissue sites as demonstrated in mouse models (4, 5) and in human peripheral blood and lungs (6, 7). We recently showed that heterogeneous populations of influenza-specific memory CD4 T cells in mice could direct rapid lung viral clearance independent of CD8 T cells and B cells but did not protect from morbidity of infection (8, 9). As circulating memory CD4 T cells reactive to pandemic influenza strains have been identified in healthy humans (10), further insights into the nature of protective memory CD4 T cells could be beneficial in optimizing this potent response.

In this study, we identify a population of influenza-specific memory CD4 T cells in lung that are noncirculating and exhibit lung-tropic migration independent of Ag specificity. We used parabiosis models to demonstrate irreversible retention of lung-tropic memory CD4 T cells in the lung and in vivo labeling to identify a noncirculating lung-resident polyclonal memory CD4 T cell population persisting after influenza infection—both exhibiting elevated CD69 and CD11a expression. Mice with memory CD4 T cells exclusively in the lung were protected from morbidity and mortality of influenza infection, with spleen-derived memory CD4 T cells affording little protection and even exacerbating mortality despite their diverse tissue residence and migration to the lung. Our results define a new class of memory CD4 T cells resident in lung tissue and reveal tissue compartmentalization as a major determining factor for protection in a key mucosal site, with important implications for targeting site-specific immunity in vaccines and immunotherapies.

BALB/c mice (8–16 wk of age; National Cancer Institute Biological Testing Branch), RAG2^−/− (BALB/c) mice (Taconic Farms, Germantown, NY), and influenza hemagglutinin (HA)-TCR transgenic mice (11) on BALB/c(Thy1.2) or BALB/c(Thy1.1) backgrounds were maintained under specific pathogen-free conditions at the University of Maryland, Columbia University, and the University of Connecticut. Protocols were approved by the institutional animal care and use committees of all institutions.

Fluorescently conjugated Abs specific for CD4, CD8, CD44, CD62L, CD69, CD90.1, CD90.2, and CD11a were purchased from BD Pharmingen (San Diego, CA). Influenza HA peptide (110–120, SFERFEIFPKE) was synthesized by the Biopolymer Laboratory (University of Maryland, Baltimore, MD).

Mice were infected intranasally with 100–500 tissue culture infectious dose units 50 (TCID₅₀) influenza virus (A/PR/8/34) for sublethal infection and 5000 TCID₅₀ PR8 influenza (2LD₅₀) for lethal infection as described (8, 9, 12). Morbidity was monitored by daily weighing and examination. Influenza virus titers were determined by TCID₅₀ assay as described (13).

HA-specific memory CD4 T cells were generated by adoptive transfer of in vitro, Ag-primed HA-TCR CD4 T cells into BALB/c(Thy1.1) or RAG2^−/− mice as previously done (4, 14, 15). Polyclonal memory CD4 T cells specific for influenza were recovered 4–8 wk after intranasal infection of BALB/c mice and quantitated by ELISPOT assay as described (8, 15).

For in vivo homing assays, HA-specific memory CD4 T cells (Thy1.1⁺) were isolated from spleens and lungs of RAG2^−/− hosts, transferred (2 × 10⁶/mouse) into BALB/c recipient mice, and recipient tissues were recovered 7 and 21 d later. For parabiosis experiments, BALB/c recipients of 2 × 10⁶ lung or spleen HA-specific memory CD4 T cells were surgically conjoined 7 d posttransfer to BALB/c mouse partners as described (16), and tissues were harvested from mouse pairs 8–21 d later.

For in vivo Ab labeling, naive or flu-immune BALB/c mice were injected intravenously with 2.5 μg PE–Cy5–conjugated anti-CD4 Ab (clone RM4-5), and after 10 min, peripheral blood was obtained and lungs perfused with PBS/500 units heparin, and cells isolated. Cells were surface or intracellularly stained with fluorochrome-conjugated Abs as described (15) and analyzed using an LSRII or FACSCanto flow cytometer (BD Biosciences, San Jose, CA) with FACSDiva (BD Biosciences) or FlowJo software (Tree Star, Ashland, OR).

Results are expressed as the mean value from individual groups ± SEM unless otherwise designated, indicated by error bars. Significance between experimental groups was determined by two-tailed Student t tests, assuming a normal distribution for all groups.

Influenza infection resulted in generation of polyclonal influenza-specific memory CD4 T cells in lung and spleen, with lung memory CD4 T cells exhibiting a slightly higher proportion of IFN-γ producers and decreased IL-2 producers compared with spleen memory CD4 T cells (Fig. 1A). To dissect the role of tissue location in the distinct functions of spleen and lung memory CD4 T cells, we generated memory CD4 T cells expressing the same TCR clonotype by adoptive transfer of in vitro-primed influenza HA-specific TCR-transgenic CD4 T cells into lymphocyte-deficient or intact mouse hosts (4, 14, 17). Similar to polyclonal populations, HA-specific memory CD4 T cells in the lung had a lower proportion of IL-2 producers and a similar frequency of IFN-γ producers compared with spleen memory CD4 T cells (Fig. 1B). These results indicate tissue-specific influences on the function of resident memory populations, independent of Ag specificity.

We hypothesized that the tissue location of memory CD4 T cells would influence their intrinsic homing capacities. Lung- and spleen-derived HA-specific memory CD4 T cells isolated as described above were transferred into unmanipulated BALB/c congenic hosts, and their resultant distribution into different tissues of recipient mice was assessed 1–3 wk later. Spleen-derived memory CD4 T cells dispersed into multiple tissues of recipient mice, including spleen, lung, liver, and lymph nodes (Fig. 1C), with the majority of spleen-derived cells recovered from the spleen, followed by reduced yet significant numbers in the lung (Fig. 1D). By contrast, lung-derived memory CD4 T cells distributed almost exclusively in the lungs of recipient mice and not significantly into other sites such as spleen, lymph node, or liver (Fig. 1C). Notably, all of the lung-derived memory CD4 T cells were recovered from the lungs of recipient mice 3 wk posttransfer (Fig. 1D), demonstrating lung tissue-tropism of this subset. Lung memory CD4 T cells localized predominantly in the tissue parenchyma with much lower numbers in the airways as quantified in bronchoalveolar lavage fluid (Supplemental Fig. 1A). Together, these results demonstrate that lung-resident memory CD4 T cells are distinct in function and migration from spleen-derived counterparts and possess internal “zip codes” enabling them to home to their tissue of residence.

The homing tropism of lung memory CD4 T cells could arise through recirculation and migration back to the lung and/or specific retention in the lung. We used a parabiosis model (16) to examine the recirculation and/or retention properties of spleen and lung memory CD4 T cells. Host mice of spleen- or lung-derived memory CD4 T cells (Thy1.1⁺) containing HA-specific memory CD4 T cells in multiple tissues or only in lungs, respectively, were surgically conjoined to a second BALB/c mouse as described (16) to create host and parabiont partner mice with shared circulations. After 8 d conjoined, T cell populations in multiple tissues of host and parabiont partner mice were recovered and analyzed. In mouse pairs containing spleen-derived memory CD4 T cells, we detected significant frequencies of HA-specific memory T cells in spleen, lung, and liver, but not lymph nodes (Fig. 2A and data not shown), with the highest overall numbers recovered from spleen and lung (data not shown). In striking contrast, mouse pairs containing lung-derived memory CD4 T cells had memory CD4 T cells exclusively in the lungs of host mice, with no dispersion into additional tissues or lung-draining lymph nodes of host or parabiont partner mice (Fig. 2A, 2B). Lung-derived memory CD4 T cells further remained in the lungs of the original host mice in long-term parabiosis experiments of 3 wk duration (Supplemental Fig. 1B), when spleen-derived memory CD4 T cells had completely dispersed and were not detected in any tissues of host or partner mice (data not shown). These results demonstrate a powerful and specific retention of lung memory CD4 T cells in the lung, independent of Ag and inflammation. These homing and retention properties of lung memory CD4 T cells are distinct from lung memory CD8 T cells, which migrate to multiple lymphoid and nonlymphoid tissue sites (9, 18) and recirculate in vivo in parabiosis studies (16), similar to our findings with spleen-derived memory CD4 T cells. Our results therefore define a new class of tissue-resident memory CD4 T cells retained within a specific compartment.

In addition to differences in CD62L expression with lung being predominantly CD62L^lo and spleen heterogeneous for CD62L expression (data not shown and Ref. 4), lung-retained HA-specific memory CD4 T cells expressed the activation marker CD69 and high levels of the integrin LFA-1 (CD11a), whereas spleen memory CD4 T cells did not express CD69 and had reduced levels of CD11a (Fig. 3A). We investigated whether subsets with different migratory capacities could be identified within endogenous lung CD4 T cells consisting of naive and memory subsets (4, 19). We adapted an in vivo Ab labeling approach that can distinguish between T cells in the lung interstitium and those accessible to the circulation (20). Administration of fluorescent anti-CD4 Ab to influenza-immune BALB/c mice resulted in >96% of blood T cells labeled (Fig. 3B, left), whereas in the perfused lung, 70% of CD4 T cells were labeled, with 20–30% protected from in vivo labeling (Fig. 3B, top). Unlabeled lung T cells exhibited an effector-memory phenotype (CD44^hi/CD62L^lo) (data not shown) with a high proportion of CD11a^hi/CD69⁺ cells (Fig. 3B, bottom), similar to the phenotype of HA-specific lung-resident memory CD4 T cells (Fig. 3A). By contrast, in vivo-labeled lung CD4 T cells were predominantly naive (70% CD44^lo/CD62L^hi; data not shown) and had reduced levels of CD11a and CD69 compared with those of the protected population (Fig. 3B, right). These results establish that the lung-resident CD69^hi/CD11a^hi memory CD4 T cell subset is represented among polyclonal populations of lung CD4 T cells. This CD69^hi/CD11a^hi phenotype is also associated with memory CD4 and CD8 T cells in intestine and skin (21, 22), suggesting similar signatures for tissue-resident T cells in other mucosal and nonlymphoid sites.

The distinct migration/retention properties of lung and spleen memory CD4 T cells enabled a novel investigation of protection by tissue-resident versus circulating memory subsets. We challenged mouse recipients of spleen or lung-derived memory CD4 T cells (“spleen-memory” and “lung-memory” recipients, respectively) intranasally with influenza PR8 (H1N1) virus (HA specificity of HA-TCR T cells) and monitored protection by weight loss morbidity and lung viral loads as described (9). After sublethal infection, naive mice and spleen-memory recipients exhibited profound and progressive weight loss of 25% by 5–6 d postinfection, whereas lung-memory recipients experienced only moderate weight loss (10%) for the entire course of infection (Fig. 4A). Importantly, lung-memory recipients exhibited more rapid and enhanced lung viral clearance compared with that of infected naive and spleen-memory recipients, with complete clearance by day 8 postinfection (Fig. 4B). Lung-memory recipients were also fully protected from lethal influenza challenge compared with naive mice (60% lethality at day 12 postinfection) and spleen-memory recipients, which experienced accelerated death at day 7 postinfection (Fig. 4C). After infection, lung-derived memory CD4 T cells maintained a biased distribution in the lung, whereas spleen-derived memory CD4 T cells were present in high frequencies in both the lung and spleen of recipient mice (Fig. 4D). These findings indicate that lung memory CD4 T cells serve as optimal in situ protectors to influenza challenge, contrasting spleen memory CD4 T cells, which exhibit low protective capacities yet extensive expansion.

Our results reveal a novel class of tissue-resident memory CD4 T cells in a key mucosal site that are specifically retained in lung tissue and mediate efficacious protection to respiratory virus infection. These findings indicate that the protective capacity of T cell memory to infections in peripheral sites is intricately linked to their tissue compartmentalization, and quantitative measurements of circulating Ag-specific T cell responses as typically assessed (23) may not reflect the quality of local tissue-specific immune responses. We propose that targeting the generation and maintenance of these tissue-resident memory populations will be critical for vaccines and immunotherapies to promote or regulate compartment-specific immune responses.

The authors have no financial conflicts of interest.