A Direct and Nonredundant Role for Thymic Stromal Lymphopoietin on Antiviral CD8 T Cell Responses in the Respiratory Mucosa

Hillary L. Shane and Kimberly D. Klonowski
J Immunol March 1, 2014, 192 (5) 2261-2270; DOI: https://doi.org/10.4049/jimmunol.1302085

Abstract

Mucosally produced thymic stromal lymphopoietin (TSLP) regulates Th2 responses by signaling to dendritic cells and CD4 T cells. Activated CD8 T cells express the TSLP receptor (TSLPR), yet a direct role for TSLP in CD8 T cell immunity in the mucosa has not been described. Because TSLP shares signaling components with IL-7, a cytokine important for the development and survival of memory CD8 T cells in systemic infection models, we hypothesized that TSLP spatially and nonredundantly supports the development of these cells in the respiratory tract. In this study, we demonstrate that influenza infection induces the early expression of TSLP by lung epithelial cells with multiple consequences. The global loss of TSLP responsiveness in TSLPR−/− mice enhanced morbidity and delayed viral clearance. Using a competitive adoptive transfer system, we demonstrate that selective loss of TSLPR signaling on antiviral CD8 T cells decreases their accumulation specifically in the respiratory tract as early as day 8 after infection, primarily due to a proliferation deficiency. Importantly, the subsequent persistence of memory cells derived from this pool was also qualitatively and quantitatively affected. In this regard, the local support of antiviral CD8 T cells by TSLP is well suited to the mucosa, where responses must be tempered to prevent excessive inflammation. Taken together, these data suggest that TSLP uniquely participates in local immunity in the respiratory tract and modulation of TSLP levels may promote long-term CD8 T cell immunity in the mucosa when other prosurvival signals are limiting.

Introduction

Mucosal surfaces, including the lung airways and the gastrointestinal tract, are major portals of Ag entry due to their large surface areas, intimate interactions with the environment, and barriers often composed of only a single layer of epithelial cells. The constant bombardment of these entry points with a variety of external stimuli, coupled with vital tissue functions that are compromised by excessive immune responses, warrants a uniquely regulated immunological microenvironment. Consequently, the mucosal immune system has adapted to respond rapidly to detrimental pathogens while maintaining tolerance against repeated nonpathogenic Ag stimulation to prevent the development of inflammatory diseases. These properties have led us and other investigators to study mucosal immune responses as unique immunological entities that when compared with systemic infection models may have different requirements for generating protective immunity and memory.

CD8 T cells are requisite for the clearance of many respiratory viral pathogens, including influenza viruses (1, 2). To date, however, most of our knowledge regarding the biology of antiviral CD8 T cell responses has been limited to models of acute, systemic infections where the tightly regulated balancing act between protection and maintenance of tissue function is not as essential. In these models, the common γ-chain (γc) cytokines play a predominant role in the antiviral CD8 T cell response, both in the effector and memory phases (3, 4). Specifically, IL-2, IL-21, IL-7, and IL-15 are known to have an influence on antiviral CD8 T cell responses, with IL-2 and IL-21 influencing early responses to infection (5–8) and IL-7 and IL-15 traditionally implicated in the formation and survival of memory CD8 T cells (4, 9, 10). However, emerging evidence suggests that many environmental factors, including the γc cytokines, relevant for optimal CD8 T cell responses in systemic antiviral immunity are either differentially regulated or disposable in mucosal systems (11–13). Indeed, data from our own laboratory has shown that memory CD8 T cells originating from a respiratory influenza infection develop and are maintained independently of IL-15, unlike those antiviral CD8 T cells derived from a systemic viral infection (10, 12). As mucosally delivered vaccines become more popular, both in concept and clinical practice, it is becoming increasingly important to understand the impact that mucosally derived factors have on the development of effective CD8 T cell responses and subsequent memory formation. One factor that is largely isolated to mucosal tissues and has the potential to influence local CD8 T cell responses is the cytokine thymic stromal lymphopoietin (TSLP).

TSLP is a γc-like cytokine that signals through a high-affinity heterodimeric receptor composed of IL-7Rα (CD127) and the specific TSLP receptor (TSLPR) (14, 15). The TSLPR is expressed on a variety of hematopoietic cell types of the innate and adaptive immune system including mast cells, dendritic cells (DCs), B cells, and T cells (16–18), as well as nonhematopoietic cells such as intestinal epithelial cells (19). Relevant to our studies, TSLP is produced constitutively by cells that constitute mucosal tissues, both in the airways and the intestinal tract (20–22), and is often elevated at these sites under inflammatory conditions such as chronic allergy and asthma (21, 23). Although epithelial cells appear to be the predominant source of TSLP in the resting mucosa, other cell types, including keratinocytes, mast cells, smooth muscle cells, and DCs, have been shown to express TSLP when exposed to a wide variety of stimuli, including TLR and NOD2 ligands, environmental stimulants, proinflammatory and Th2 cytokines, and viruses (24). Because TSLP production is enriched at mucosal surfaces, particularly following inflammatory or viral stimuli, TSLP signaling may uniquely modulate immune responses in these sites.

Most research on TSLP has focused on the cytokine’s effect on CD4 T cells, the development of Th2 immune responses, and asthma, leaving the influence of TSLP on the CD8 T cell response to infection less well explored. The TSLPR is expressed on naive murine CD8 T cells at low levels (18) and is undetectable on naive human CD8 T cells (25), limiting the ability of TSLP to act directly on these cells. However, the TSLPR is transiently upregulated following TCR stimulation in both mice and humans (18, 25), enhancing the potential for TSLP to act directly on activated CD8 T cells. Indeed, provision of TSLP to CD8 T cells activated by anti-CD3/anti-CD28 in vitro induces STAT5 phosphorylation, the upregulation of Bcl-2, and increased survival, although to a much lesser extent than providing IL-7 (18). Importantly, cells destined to become memory CD8 T cells preferentially express CD127 (26). As the receptors for TSLP and IL-7 both share CD127 and some downstream signaling components (27), it is possible that the two cytokines may have some overlapping and/or nonredundant functions related to memory cell survival. In summary, these data suggest that TSLP has the capability to act directly on CD8 T cells; however, to date, investigators have yet to define a direct role for TSLP on Ag-specific CD8 T cell responses independent of the effects of the cytokine on secondary players (i.e., DCs or CD4 T cells) participating in the immune response.

In this study we sought to determine whether mucosally derived TSLP acts directly on CD8 T cells after influenza infection and influences their response to infection and/or subsequent development into specific memory cell pools in a way distinct from other cytokines. We show that TSLP is produced locally following influenza virus infection and positively regulates the antiviral response. Importantly, TSLP acts directly on Ag-specific CD8 T cells in the respiratory tract in a manner that increases their proliferation and persistence into later stages of the immune response. To our knowledge, our study implicates a newly defined role for TSLP acting on Ag-specific CD8 T cells responding to an infection and adds to the emerging story designating unique roles for cytokines in the context of mucosal immune responses.

Materials and Methods

Mice and viruses

C57BL/6 mice were purchased from Charles River (Wilmington, MA) through the National Cancer Institute program, and TSLPR−/− mice (28) were provided by Dr. Steve Ziegler (Benaroya Research Institute, Seattle, WA). C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-I) mice were provided by Dr. Leo Lefrançois (University of Connecticut, Farmington, CT) and maintained on a CD45.1 Rag−/− background. These mice were bred in house with CD45.2 TSLPR−/− mice to produce CD45.2 and CD45.1/CD45.2 TSLPR−/− OT-I mice on a Rag−/− background. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Georgia. The influenza virus A/HK-x31 (x31; H3N2) was provided by Dr. S. Mark Tompkins (University of Georgia, Athens, GA) whereas the recombinant x31-OVA expressing the CD8 H2-Kb–restricted SIINFEKL epitope was provided by Dr. Peter Dougherty (St. Jude Children’s Research Hospital, Memphis, TN).

Influenza infections

For in vitro experiments, mouse lung epithelial (MLE)-15 cells (29) were grown in 12-well plates in HITES medium (30) supplemented with 4% FBS (growth media) and either mock or x31 infected with a multiplicity of infection (MOI) of 50% (0.5 MOI) for 1 h at 37°C in growth media. Following infection, cells were washed with PBS and then cultured in growth media until cells were harvested. For in vivo experiments, age- and sex-matched anesthetized animals were infected intranasally (i.n.) with 103 PFU x31 or x31-OVA in 50 μl PBS. Mock-infected animals received 50 μl PBS i.n.

Quantitative RT-PCR

Cells or whole tissues were collected in RNAlater (Qiagen, Valencia, CA) or PrepProtect (Miltenyi Biotec, Auburn, CA) and stored at −80°C until processing. RNA was purified from the samples using the RNeasy Plus Mini kit (Qiagen). Reverse transcriptions were performed using the high-capacity cDNA reverse transcription kit from Applied Biosystems (Foster City, CA). Quantitative PCR assays were prepared using the ABI TaqMan Gene Expression Master Mix from ABI 7500 real-sime PCR System and TSLP-FAM (Mm01157588_m1) and 18s-VIC (no. 4319413E) assays in a multiplex reaction assessed on an ABI 7500 real-time PCR system (Applied Biosystems). Thermal cycling conditions were 2 min at 50°C, 10 min at 95°C, and 40 cycles of denaturation (95°C for 15 s) and annealing (60°C for 60 s). Samples were analyzed in triplicate, normalized against 18S, and expressed relative to mock-infected animals. The results are expressed as relative quantity over mock-infected control samples determined by the ΔΔ cycle threshold method with analysis performed on the ABI 7500 system SDS software version 1.3.1.

Plaque assays

Plaque assays were performed as previously described (31). Briefly, whole lungs from infected mice were isolated, weighed, and homogenized using a TissueLyser (Qiagen, Hilden, Germany). Serial dilutions of 10% homogenate were made in dilution media (1× MEM, 1 μg/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone–treated trypsin) and incubated for 1 h atop confluent monolayers of Madin–Darby canine kidney cells grown in 12-well plates for 1 h at 37°C. Following infection, cell layers were washed with PBS and overlaid with MEM containing 1.2% Avicel microcrystalline cellulose (FMC BioPolymer, Philadelphia, PA), 0.04 M HEPES, 0.02 mM l-glutamine, 0.15% NaHCO3 (w/v), and 1 μg/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone–treated trypsin. After 72 h at 37°C, the overlay was removed and the cells were washed with PBS, fixed by incubation with cold methanol/acetone (60:40%), and stained with crystal violet. Plaques were counted and PFUs per milligram of lung tissue determined.

Tissue preparation

Single-cell suspensions from tissues were obtained as previously described (12). Briefly, cells from the lung airways were obtained by means of bronchoalveolar lavage (BAL) in which the trachea was intubated and 1 ml PBS was introduced and recovered from the lung airway four times. Following BAL collection, cells were isolated from the lung parenchyma after first perfusing the lungs with ∼10 ml PBS/heparin. The perfused lungs were excised, minced, and incubated with 1.25 mM EDTA at 37°C for 30 min followed by a 1-h incubation with 150 U/ml collagenase (Life Technologies, Grand Island, NY). After passage through cell strainers, lymphocytes were resuspended in 44% Percoll, underlaid with 67% Percoll, centrifuged, and the cellular interface was collected. Lymph nodes and the splenic tissues were mechanically disrupted and then passed through a cell strainer. Erythrocytes were depleted from the spleens using Tris-buffered ammonium chloride. Cell numbers were determined using a Z2 Coulter particle counter (Beckman Coulter, Fullerton, CA).

Flow cytometry

The influenza nuclear protein (NP) MHC class I (H-2Db/ASNENMETM) tetramer was generated at the National Institute of Allergy and Infectious Diseases Tetramer Facility (Emory University, Atlanta, GA). Staining was carried out at room temperature for 1 h in conjunction with other surface staining mAbs: PerCP/Cy5.5-conjugated anti-CD8α or anti-CD44, FITC-conjugated anti-CD11a or anti-CD122, PE-conjugated anti-CD127 or anti-CD43, allophycocyanin/Cy7-conjugated anti-CD62L or anti-CD8α, and PE/Cy7-conjugated anti-KLRG1 or anti-CD27 (all from eBioscience, San Diego, CA). When tetramer was not used, cells were surface stained for 20 min at 4°C. Data were acquired using an LSR II with FACSDiva software (BD Biosciences, San Jose, CA) and analysis of data was performed using FlowJo software (Tree Star, Ashland, OR). All samples were gated on single cells prior to subsequent gating and analysis.

CD4 T cell IFN-γ production assay

Following isolation, lymphocytes were incubated at 37°C for 5 h with or without the x31-derived hemagglutinin (HA) (195–209) (YVQASGRVTVSTRRS) peptide (AnaSpec, Fremont, CA) in the presence of GolgiStop (BD Pharmingen, San Diego, CA). Naive splenocytes (1 × 106) were added to the lymphocyte populations isolated from the BAL as an Ag-presenting population. Following the stimulation period the cells were extracellularly stained with anti-CD8, anti-CD4, and anti-CD44 Abs (eBioscience, San Diego, CA) for 20 min at 4°C, fixed in 2% paraformaldehyde overnight, permeabilized using Perm/Wash buffer (BD Biosciences, San Diego, CA), and intracellularly stained using FITC-conjugated anti–IFN-γ (BD Pharmingen) for 30 min at 4°C. Samples were analyzed by flow cytometry as described above.

Proliferation/death assays

At 6 d postinfection (p.i.) mice were injected i.p. with 1 mg BrdU solution (BD Pharmingen). Twenty-four hours after injection, mice were sacrificed and tissues were collected. Isolated lymphocytes were first surface stained as previously described and subsequently stained intracellularly using anti-BrdU mAb conjugated to allophycocyanin (BD Pharmingen). To assay cell death, lymphocytes isolated at the indicated times were first surface stained with the appropriated identifying Abs and then incubated with annexin V–PE and 7-aminoactinomycin D (7-AAD) viability staining solution (eBioscience) and analyzed via flow cytometry.

Competitive adoptive transfers

Splenocytes isolated from CD45.1 OT-I mice and CD45.2 or CD45.1/CD45.2 TSLPR−/− OT-I mice were counted, resuspended in PBS at 1000 cells/100 μl, and injected at a 1:1 ratio i.v. into congenically distinct (CD45.1/CD45.2 or CD45.2) recipient mice. Twenty-four hours after transfer, mice were infected i.n. with x31-OVA. Donor cells were detected by flow cytometry using mAb from eBioscience for the appropriate anti-CD45 molecule (PE/Cy7-conjugated anti-CD45.1, allophycocyanin-conjugated anti-CD45.2), along with stains for phenotyping of the donor populations (allophycocyanin/Cy7-conjugated anti-CD8α or anti-CD62L, PE-conjugated anti-Vα2, FITC-conjugated anti-CD44, and PerCP/Cy5.5-conjugated anti-CD127.

Statistical analysis

Statistical analysis was carried out using Prism 5 software (GraphPad Software). Significance was determined when the p value was <0.05 and is indicated, along with the type of analysis used, in the figure legends.

Results

In vitro and in vivo induction of TSLP mRNA following influenza infection

Following inhalation, influenza virions preferentially infect respiratory epithelial cells, which alert the immune system to infection via activation of TLR3 and TLR7 and RIG-I pattern recognition receptors (32, 33). Studies have demonstrated that stimulation of TLR3 using dsRNA can induce the expression of TSLP in human airway (16) and bronchial epithelial cells (34). Moreover, TSLP expression was also enhanced following infections with the respiratory pathogens rhinovirus and respiratory syncytial virus in human and rat airway epithelial cells, respectively (35). Recently, it has also been shown that infection with the highly pathogenic strain of influenza A virus, Puerto Rico/8, can induce the production of TSLP mRNA in the lungs and trachea of mice (36), although data are conflicting as to whether this affects the anti-influenza CD8 T cell response, either directly or indirectly, and to what extent (36, 37).

In this study we assessed the role that TSLP plays on the immune response to the influenza virus A/HK-x31 (x31; H3N2). This virus closely mimics seasonal influenza infections and attenuated vaccines, as it is much less pathogenic in mice, even at high doses. We first used an in vitro culture system to determine whether x31 infection could elicit TSLP mRNA expression because TSLP protein expression has been difficult to reliably detect using ELISA-based methods. The mouse lung epithelial cell line MLE-15 was infected with x31 or mock infected with PBS. Following infection, the cells were maintained in culture for the indicated times until they were harvested and real-time quantitative PCR (RT-qPCR) for murine TSLP mRNA was performed. Infected samples were directly compared with mock-infected controls incubated in the same culture conditions for corresponding amounts of time. TSLP mRNA was induced in MLE-15 cells after influenza infection as early as 12 h p.i. and expression remained elevated until as late as 72 h p.i. (Fig. 1, top left), at which point the experiment was terminated owing to increasing levels of epithelial cell death. Although these data demonstrate that x31 infection can elicit the production of TSLP by lung epithelial cells in vitro, it was unclear whether analogous infection of mice in vivo could also induce TSLP expression.

FIGURE 1.
FIGURE 1.

Influenza infection induces the expression of TSLP mRNA. The murine lung epithelial cell line (MLE-15) was infected with an MOI of 0.5 x31 and cultured for the indicated times after infection before cells were harvested, RNA was extracted, and RT-qPCR was performed. For in vivo analysis, C57BL/6 mice were infected with 1000 PFU x31 or mock infected with PBS i.n. Tissues were collected at the indicated times after infection and TSLP mRNA was quantified using RT-qPCR. Data were normalized using an endogenous control and are displayed as relative quantification over mock-infected controls (1 or reference [Ref]) for each indicated time point, as determined by the ΔΔ cycle threshold method. Values are shown as mean relative quantification (RQ) ± SEM (n = 3 samples/group). Data are representative of two independent experiments.

To test whether TSLP is produced following x31 infection in vivo, mice were infected i.n. with the virus or mock infected with PBS. At the indicated times after infection, BAL fluid, lungs, the lung draining mediastinal lymph nodes (MdLNs), and spleens from infected and mock-infected animals were harvested and TSLP mRNA levels were quantified via RT-qPCR. During the course of infection, TSLP levels in the spleen (Fig. 1, top right) were low and did not increase above the reference levels observed after a mock infection. In contrast, TSLP mRNA production was increased in the lung by 24 h after x31 infection (Fig. 1, bottom left). These levels remained elevated until at least day 10 p.i., which corresponds to the peak of the anti-influenza CD8 T cell response in the respiratory tract. TSLP levels in the lung subsequently returned to baseline by day 15 p.i. when most anti-influenza CD8 T cells are contracting and some are transitioning to memory. TSLP had similar fold increases in expression in the MdLN as in the lung following infection with x31, peaking at 2 d p.i. (Fig. 1, bottom right), indicating that TSLP is present at the site of T cell priming and may act on these influenza-specific CD8 T cells during their initial activation. Because the timing of TSLP expression in the draining lymph nodes correlates well with the arrival of migratory DCs to this site (38), and TLR-activated DCs have been shown to produce TSLP (39, 40), it is possible that this cell population confers TSLP expression at the site of T cell priming. However, note that constitutive TSLP mRNA levels in the lung were ∼10-fold higher than those observed in the MdLN, which had little to no TSLP expression in mock-infected animals (data not shown). Therefore, although fold induction was similar between lung and MdLN at the peak of expression (∼4-fold), the levels of TSLP mRNA were actually much higher in the lung both before and after influenza infection. These data indicate that although some migratory lymphoid cells (perhaps DCs) are able to induce TSLP expression in sites within close proximity to the respiratory tract, the lung is the main TSLP source after influenza infection. TSLP mRNA was not detected in the cells obtained from the BAL (data not shown), further confirming that nonlymphoid cells predominantly contribute to TSLP production in situ. Taken together, these data demonstrate that respiratory infection with the x31 influenza virus evokes the local production of TSLP, which is likely the result of the viral infection of the epithelial cells themselves as opposed to the highly inflammatory environment associated with more immunopathogenic influenza viruses. Moreover, the location and kinetics of TSLP expression following influenza infection suggest that TSLP can act on CD8 T cells during their initial priming event in the MdLNs and later upon their arrival as effector cells at the infection site.

Global loss of TSLPR signaling increases morbidity in mice after influenza infection

Once it was established that local TSLP expression was induced by influenza infection, we wanted to determine whether the global loss of TSLP signaling would impact the overall anti-influenza response. Toward this end, age- and sex-matched TSLPR−/− mice and wild-type (WT) C57BL/6 controls were infected with a sublethal dose of x31 and overall morbidity, viral burdens, and adaptive immune responses were measured. As indicated in Fig. 2A, infection with the mouse-adapted x31 strain of influenza results in overall low morbidity in WT mice, which lose negligible weight during the course of infection. In contrast, TSLPR−/− mice lost significantly more weight than did WT controls beginning as early as day 2 p.i. and continued until about a week after infection, with starting body weight recovered by ∼8 d p.i. (Fig. 2A). The early and sustained weight loss in TSLPR-null mice may be the result of the inability of these animals to repair and maintain epithelial cell tight junctions (41), which were initially disrupted after influenza infection (42). In addition to the role that TSLP may be playing in enhancing the integrity of the epithelial barrier, the increased morbidity seen in the TSLPR−/− mice could result from increased viral titers, immunopathology, or an inability to heal as well as WT mice (19).

FIGURE 2.
FIGURE 2.

TSLPR−/− mice develop more severe disease than do WT mice. (A) WT B6 mice (n = 10) and TSLPR−/− mice (n = 9) were infected i.n. with 1000 PFU x31 and weighed daily. Data are shown as mean percentage change in body weight over time ± SEM. Overall significance in weight change between WT and TSLPR−/− mice was assessed using a two-way ANOVA test (p = 0.0017). Significance between groups at individual time points was assessed using a two-tailed Student t test. Data are representative of two independent experiments. Data are representative of two independent experiments. *p ≤ 0.05, **p < 0.01, ***p < 0.001. (B) WT and TSLPR−/− mice were infected i.n. with 1000 PFU x31. Viral titers were measured by plaque assay at days 2, 3, 4, 6 (left), and 8 (right) p.i. and graphed as PFU/mg lung tissue ± SEM (n = 3 mice/group/d). Data are representative of two independent experiments. (C) Lymphocytes isolated from the BAL, lung, and spleen of WT and TSLPR−/− mice (n = 3/group) at 10 d after x31 infection were stimulated ex vivo in the presence or absence of a class II–restricted HA peptide, and CD4 T cells were assessed for IFN-γ production. The top panels show representative IFN-γ staining in CD4+ (CD44hi) lymphocytes. The bottom panel shows the quantification of these data. Data are representative of two independent experiments.

To test whether TSLPR−/− mice have a deficiency in their ability to clear influenza infection as effectively as their WT counterparts, both groups of mice were infected i.n. with x31, lungs were harvested at the indicated times following infection, and viral titers were assessed by plaque assay (Fig. 2B). Early after infection and at the peak of viral replication (days 2–3), we did not observe any difference in viral titers between WT and TSLPR−/− mice. However, at day 4 and particularly by day 6 p.i., TSLPR−/− mice harbored higher viral titers in their lungs. TSLPR−/− mice were unable to fully clear the virus by day 8 p.i. whereas WT mice had completely resolved the infection (Fig. 2B). Data from later time points show that TSLPR−/− mice eventually clear virus with a 2 d delay over WT mice or by day 10 p.i. (data not shown). Taken together, these data indicate that TSLPR−/− mice were not more susceptible to influenza infection, as early viral titers and peak viral titers were similar, but instead they had a defect in their ability to clear the virus as rapidly as WT mice.

The prolonged viral burden in the TSLPR−/− mice could be the result of a defective or delayed adaptive immune response. We first analyzed the CD4 T cell response in WT versus TSLPR−/− mice, as TSLP is well known to influence CD4 T cell polarization both directly (43, 44) and through interactions with DCs (45). CD4 T cells isolated from the BAL, lung, and spleen of WT and TSLPR−/− mice 10 d p.i. were equally competent in their ability to produce IFN-γ after stimulation with the x31 influenza CD4 HA epitope (Fig. 2C). Limited data suggest that TSLP can modulate Ab responses (46); however, we did not expect the anti-influenza IgG2a titers, which correlate with influenza virus clearance (47), to be significant early enough to impact viral clearance prior to day 10 p.i. Indeed, compared with x31 immune animals there were no detectable levels of these Abs in either WT or TSLPR−/− sera 9 d p.i. (data not shown).

Because CD8 T cells are requisite for efficient and complete clearance of influenza virus (1, 2), their localization and activity in response to influenza infection coincides with the timing of respiratory TSLP expression (48) (Fig. 1), and because TSLPR is expressed on activated CD8 T cells (18, 25), we reasoned that TSLP may be acting on CD8 T cells at the site of infection. To evaluate the role that TSLP may play on anti-influenza CD8 T cell responses, we used an MHC class I tetramer loaded with the immunodominant epitope of the influenza NP H-2Db/ASNENMETM. Using this reagent, we assessed the frequency of influenza-specific CD8 T cells present in both the lymphoid tissues (spleen and lymph nodes) and peripheral effector sites (lung and BAL) at effector and memory phases of the anti-influenza response in WT and TSLPR−/− mice (Fig. 3). We did not observe any difference in the overall frequencies of NP-specific CD8 T cells isolated from the assayed lymphoid and nonlymphoid tissues at the peak of infection (10 d p.i) or during time frames consistent with the development of early (32 d p.i.) or late (115 d p.i.) memory CD8 T cells. Additionally, there were no significant differences in the total number of NP-specific CD8 T cells during the period assayed (data not shown). We also failed to observe any difference between the two groups regarding the phenotype of NP-specific cells recovered after infection in terms of CD127, KLRG1, CD62L, CD122, CD27, and CD43 expression (data not shown).

FIGURE 3.
FIGURE 3.

TSLPR−/− mice harbor similar frequencies of influenza-specific CD8 T cells as WT mice. Lymphocytes were isolated from the indicated tissues of x31-infected animals and analyzed by flow cytometry for tetramer reactivity at 10, 35, and 115 d p.i. (A) Representative dot plots from the BAL, lung, spleen, MdLN, and nondraining inguinal LN (ILN) of WT and TSLPR−/− mice at day 10 p.i. Cells were first gated on total CD8+ lymphocytes and analyzed for CD11a expression and tetramer reactivity. (B) Average percentage NP-tetramer+ of CD8 T cells isolated from indicated tissues at days 10, 32, and 115 p.i. in WT and TSLPR−/− animals. Data are shown as the mean percentage of tetramer+ cells of the CD8 T cells ± SEM (n = 3 mice/group) and are representative of two independent experiments. Data were analyzed for significance using a two-tailed Student t test. No significant differences were found.

TSLP can act directly on CD8 T cells at the site of infection, influencing their proliferative and developmental fate

Although we did not observe any difference in the frequency of influenza-specific CD8 T cells recovered between WT and TSLPR−/− mice, it was still quite possible that TSLP functionally participated in the normal CD8 T cell response to respiratory infection. Our inability to detect any CD8 T cell deficiencies in the TSLPR−/− mouse could be due to cytokine redundancy in which those cytokines eliciting similar functions (similar to IL-7) could functionally compensate and mask the consequences of loss of TSLP alone. Furthermore, nonredundant roles of cytokines can be difficult to discern in vivo where disparities in the extent of infection and inflammation between individual animals and/or strains may conceal subtle differences in CD8 T cell responses. Thus, we modified our experimental system to better determine the role that TSLP plays on the anti-influenza CD8 T cell response directly, exclusively, and with greater sensitivity.

To elucidate the direct and individual contribution of TSLP to the anti-influenza CD8 T cell response, we used a competitive adoptive transfer system. In this system, the response of TSLPR-deficient and -sufficient CD8 T cells of identical specificity is assayed within the same host. By design, this experimental system will reveal the functional consequences of the individual loss of TSLP signaling, even when compensatory pathways are present, as fully competent WT Ag-specific CD8 cells could have a selective advantage over TSLP signaling–deficient cells. Moreover, any secondary effects resulting from loss of TSLP signaling are excluded from the analysis. We therefore incorporated the competitive adoptive transfer scheme outlined in Fig. 4A, in which we adoptively transferred 1000 congenically mismatched TSLPR−/− OT-I cells and TSLPR+/+ OT-I cells (WT OT-I) into congenically unique recipients. Using this method, we isolated the effects of TSLP signaling deficiency to the T cells themselves, independent of the indirect effects of TSLP on CD8 T cells via DCs or CD4 T cells.

FIGURE 4.
FIGURE 4.

TSLPR−/− OT-I cells are less prevalent at the site of infection than are WT OT-I cells following influenza infection. (A) Competitive adoptive transfer scheme; congenically mismatched WT OT-I and TSLPR−/− OT-I cells (1000 each) were adoptively transferred via tail vein injection into congenically distinct WT mice that were infected 24 h later i.n. with 1000 PFU x31-OVA . (B) Representative flow from the indicated tissues at days 8 and 50 p.i. Single-cell lymphocyte populations were first gated on double-positive Vα2+CD44hi cells. (C) Frequencies of WT and TSLPR−/− OT-I cells at days 8 and 50 following infection as assessed by flow cytometry. Data are shown as pooled samples from three identical experiments and expressed as a ratio of TSLPR−/− OT-I cells/WT OT-I cells. Significance was determined using a one-sample t test against a theoretical mean of 1. **p < 0.01, ***p < 0.001.

To test the hypothesis that the direct loss of TSLP signaling on CD8 T cells impacts their response to respiratory viral infection, we i.n. infected our recipient mice harboring the OT-I chimeras with x31-OVA and monitored the frequencies of the specific OT-I subsets over time using the appropriate combination of CD45 Abs. Comparisons of the ratio of TSLPR−/− to WT OT-I cells at the proliferative peak of the anti-OVA CD8 T cell response (day 8 p.i.) demonstrated that WT OT-I cells preferentially accumulated proximal to the site of infection (BAL, lung, and MdLN) compared with TSLPR−/− OT-I cells (Fig. 4B) although there was little difference in the accumulation of either genotype of Ag-specific effector CD8 T cells at sites distal to the infection (spleen and inguinal lymph nodes). Importantly, the inability of TSLPR-deficient OT-I cells to accumulate in the respiratory tract was maintained and exacerbated into the development of memory as observed at day 50 p.i., and this trend also continued until day 125 p.i., although donor populations became more difficult to find (data not shown). These data suggest that the early defect in the TSLPR-deficient CD8 T cell response in the respiratory tract is maintained and numerically affects the resultant population of memory CD8 T cells. The result is particularly important, as maintenance of CD8 T cells in the respiratory tract is requisite for prolonged CD8 T cell–based heterosubtypic immunity to influenza infection (49, 50).

To determine whether TSLP signaling qualitatively influences the development of memory cells, we analyzed the transferred OT-I cells for the expression of CD127 and CD62L. CD127 is used as a marker to delineate the precursors of bona fide memory cells (26), and IL-7 signaling via CD127 is important for their long-term survival after systemic infection (9, 51). Overall, no significant differences were observed in the CD127 expression between WT or TSLPR−/− OT-I CD8 T cells (Fig. 5A). However, differences were seen in the levels of CD127 on a per cell basis (as measured by mean fluorescence intensity) between the OT-I cells derived from lymphoid (Spl and LNs) versus peripheral (lung and BAL) sites, with the latter showing decreased levels of CD127 expression. Interestingly, we observed a difference in the expression of CD62L between the OT-I groups 50 d p.i. (Fig. 5B). The OT-I memory cells deficient in TSLP signaling harbored a greater proportion of CD62L+ cells in the BAL, lung, MdLN, and the spleen compared with their WT counterparts (Fig. 5B). The selectin CD62L confers lymph node homing potential, and it is used to distinguish populations of memory cells as being either central memory cells (CD62L+) or effector memory cells (CD62L−). In this context, TSLP expression may influence the development of memory cells, with either early or sustained TSLP signaling throughout the transition to memory promoting an effector memory phenotype typical of influenza-specific CD8 T cells derived from mucosal sites. Taken together, these experiments demonstrate that following respiratory infection with influenza virus, TSLP signals directly to TSLPR-competent Ag-specific CD8 T cells in the respiratory tract. As a result, the number of Ag-specific cells at the site of infection is increased at the peak of the CD8 T cell response, which carries over into the resulting CD8 memory cell pool. Moreover, TSLP may concomitantly modify the phenotype of the memory populations as assayed by differential CD62L expression.

FIGURE 5.
FIGURE 5.

TSLPR−/− OT-I cells express higher levels of CD62L than do WT-OT-I cells. (A) Mean fluorescence intensity (MFI) was determined on the transferred populations of cells for staining against CD127 at day 50 p.i. Data are representative of three independent experiments (n = 6). (B) CD62L expression was analyzed on the populations of adoptively transferred cells at day 50 p.i. Data are pooled from three independent experiments and displayed as frequency of CD62L high cells among total WT and TSLPR−/− OT-I donors (with these populations within individual recipients connected by line). Data are shown as pooled samples from three identical experiments. Statistical significance was determined using a two-tailed Student t test comparing the mean frequency of CD62Lhigh OT-I cells between groups. *p < 0.05, **p < 0.01.

TSLP can affect the magnitude of the CD8 T cell pool at the site of infection in several ways: by modulating migration into the tissue, by promoting in situ proliferation, and/or by enhancing survival of the cells. Although it has been reported that TSLPR−/− cells express lower levels of the inflammatory chemokine receptor CXCR3 (37), TSLPR−/− OT-I cells were able to accumulate in the respiratory tract (Fig. 4), indicating to us that loss of TSLP did not impact migration to the mucosa. Rochman and Leonard (18) showed that TSLP positively affected the survival of CD8 T cells under homeostatic conditions via the upregulation of Bcl-2. Conversely, Akamatsu et al. (25) showed that TSLP enhanced the proliferation of ex vivo–stimulated human CMV-specific CD8 T cells. In our adoptive transfer system, infection of recipient animals harboring WT OT-I CD8 T cells with x31-OVA resulted in a dramatic increase of these Ag-specific CD8 T cells starting at day 7 p.i. and peaking sharply at day 8 p.i., before decreasing, once again quite dramatically, by day 9 p.i. (Fig. 6A). This curve indicates a period of rapid proliferation prior to the peak of OT-I CD8 T cell response that is followed by rapid death of these cells. Therefore, to gain a better understanding of how TSLP signaling influences Ag-specific CD8 T cells in the respiratory tract, we assayed both proliferation and survival of the adoptively transferred populations of cells at physiologically relevant times surrounding the peak of the response. To assay cellular proliferation, recipient mice were injected i.p. with the thymidine analog BrdU at 6 d p.i. Twenty-four hours later (7 d p.i.) lymphocytes were isolated from the indicated tissues and surface stained for their identifying congenic markers and intracellular BrdU. Although varying levels of proliferation were observed among the recipient mice, consistent differences were observed between the level of proliferation of OT-I cells derived from the WT and TSLPR−/− backgrounds within a single recipient mouse. Within the respiratory tract, TSLPR−/− OT-I cells proliferated ∼10% less than their WT OT-I counterparts (Fig. 6B, 6C). These results were significant specifically at the site of infection (BAL and lung), where mucosally produced TSLP could directly act on respondent Ag-specific effector CD8 T cells. At day 7 p.i. we also observed that the cell cycle marker Ki-67 was expressed in a lower frequency of TSLPR−/− OT-I cells compared with WT OT-I cells (data not shown), further indicating that TSLP signaling on CD8 T cells directly leads to increased levels of Ag-specific CD8 T cell proliferation. Surprisingly, and in contrast to published findings that TSLP regulates the survival of activated CD8 T cells both in vitro and in vivo (18), we did not observe any differences in the rate of cell death between WT and TSLPR−/− OT-I cells as measured by staining with 7-AAD and annexin V at days 9 and 10 p.i. (Fig. 6D). These data indicate, to our knowledge, a previously undefined role for TSLP, where the cytokine produced in the respiratory mucosa acts directly on responding Ag-specific CD8 T cells to increase their local proliferation and establishment as a pool of memory cell precursors at this site.

FIGURE 6.
FIGURE 6.

TSLPR−/− OT-I cells proliferate less following influenza infection than do WT OT-I cells. (A) Two thousand CD45.1 OT-I cells were transferred into CD45.2 recipient mice and infected i.n. with HKx31-OVA 24 h later. Number of donor CD8 T cells in the BAL, lung, and spleen were quantified using flow cytometry and displayed as mean ± SEM (n = 4 mice/group). (B and C) Congenically distinct TSLPR−/− OT-I and WT OT-I cells (1000 each) were transferred into recipient mice that were infected i.n. with x31-OVA 24 h following transfer. BrdU (100 μg) was administered i.p. at 6 d p.i. (24 h prior to sacrifice) and BrdU incorporation was assessed at 7 d p.i. by intracellular staining followed by flow cytometric analysis. (B) The left panel depicts representative BrdU staining in either the WT or TSLPR−/− OT-I pools activated in the same animal. BrdU incorporation for the individual OT-I pools is quantified for all tissues analyzed on the right. Data are shown as percentage of BrdU+ cells where each set of connected points represents the transferred populations of OT-I cells found within the same recipient mouse. Differences in the level of BrdU incorporation between the WT and TSLPR−/− OT-Is are depicted for the respiratory tract only (C). Significance in (B) and (C) was tested for using a paired Student t test. Data shown are representative of three experimental repeats. *p < 0.05. (D) Cell death was measured by Ab staining for annexin V and 7-AAD in the indicated tissues at 9 and 10 d p.i.; each set of connected dots represents the transferred OT-I populations within the recipient mouse. Data shown are representative of two experimental repeats. Significance was tested for using a paired Student t test and no significant differences were found.

Discussion

Mucosal surfaces harbor unique and specialized immunological niches that are tightly regulated to promote immunity while causing minimal immunopathology. Mucosal environments employ many regulatory mechanisms, both constitutively and when faced with inflammatory stimuli, to maintain their vital tissue function. Cytokines classified as anti-inflammatory (TGF-β and IL-10) or Th2 biasing (IL-4, TSLP, and the alarmin IL-33) are integral in maintaining mucosal tissue integrity. Dysregulation of many of these cytokines results in the development of inflammatory bowel or allergic airway diseases (52, 53), highlighting the importance of these cytokines in immune homeostasis at barrier sites. Relevant to our work, studies have demonstrated that respiratory viral infections can enhance levels of IL-33 (54) and TSLP (35), and disruption of these cytokine networks results in poor immunological outcomes in response to these pathogens (55). Whereas in many cases this is directly related to defective barrier function (45), direct modulation of immune effectors by these cytokines could also impact immunity.

With growing interest in developing mucosal vaccines, particularly those targeting CD8 T cells (56, 57), there is a need to gain a deeper understanding of how cytokines influence the development and maintenance of memory CD8 T cells at these sites. It is known that during the course of the CD8 T cell response to infection, diametric signals exist to positively direct antiviral CD8 T cells toward a memory versus short-lived effector cell fate. One way to discriminate memory cell potential is through the expression of IL-7Rα, which imparts a survival advantage to this pool of cells after IL-7 encounter (9). However, evidence suggests that redundant mechanisms also exist to regulate memory cell fate (58) and that different signaling pathways may regulate memory CD8 T cell development in systemic versus mucosal infection (12).

Our original hypothesis was that mucosally derived TSLP, which shares common signaling pathways with IL-7 and binds to a receptor also containing IL-7Rα, participates as an alternative and nonredundant pathway for memory CD8 T cell development in mucosal sites. However, we observed that TSLP participates much earlier in the anti-influenza CD8 T cell response by promoting the local proliferation of Ag-specific effector CD8 T cells (Fig. 6B) that not only temporally increases their number but also those memory cells derived from this pool (Fig. 4B). This proliferative role of TSLP was only apparent in the competitive adoptive transfer system where competition between TSLPR-deficient and WT CD8 T cells for identical resources revealed this distinct and nonredundant function for TSLP. The proliferative role for TSLP was also only observed in the BAL and lung (Fig. 6B), highlighting the relevance of this cytokine in driving CD8 T cell division in situ. Whether low-level constitutive TSLP expression in the respiratory tract is responsible for any periodic homeostatic proliferation of memory T cells over time is still unknown. Interestingly, however, the proliferative function of TSLP in the context of influenza infection is independent of alterations in survival (Fig. 6D), unlike the results from previous studies that assessed the role of TSLP in homeostatic conditions (18). These data would suggest that perhaps TSLP signaling has contextual effects on CD8 T cell responses dependent on location, signaling thresholds, and cytokine/receptor expression levels, many of which are different under inflammatory and homeostatic conditions.

An important aspect of our study was that TSLP affected local effector cell proliferation, and ablation of TSLPR signaling did not completely block the development and maintenance of memory CD8 T cells, likely due to intact cytokine networks. IL-2 and IL-7 support the early division and survival outside of the respiratory tract and are available to effector CD8 T cells early after activation (3).Whereas IL-7 in particular is superior compared with TSLP in providing survival and/or proliferation signals to CD8 T cells (18, 37), naive T cells trafficking between secondary lymphoid tissues compete for IL-7 survival signals (59). As a consequence of this competition, peripheral memory cells express higher levels of CD127 on a per cell basis compared with their naive counterparts (26). Mucosal (versus systemic) memory CD8 T cells express less CD127 (Fig. 5B) (11), but they do not compete with naive cells for TSLP in the respiratory tract, such that even limited CD127 expression could fully support TSLP signaling in situ. Likewise, it is unclear whether cytokine compartmentalization results in a greater dependence on TSLP once effector CD8 T cells enter the respiratory tract. Although posttranslational regulation of TSLP is unknown, IL-7 bioavailability is carefully regulated in vivo via selective binding to heparin sulfate moieties on basement membranes whose composition may differ in the lung (60). Lack of reliable assessment of IL-7 and TSLP protein levels by standard methods have prevented testing the hypothesis that cytokine bioavailability limits respiratory CD8 effector T cell proliferation. However, the expression of TSLP mRNA clearly supports a spatial and temporal focus that could influence anti-influenza effector CD8 T cells that seed the respiratory tract and differentiate into memory cells in situ. Moreover, the inferior TSLP-driven proliferative signal in the respiratory tract could account for the limited survival of airway-resident effector/memory cells observed in influenza infection models (50).

Interestingly, loss of TSLP signaling resulted in the increased expression of CD62L on the Ag-specific OT-I cells in the respiratory tract compared with the WT OT-I cells at 50 d p.i. (Fig. 5B). Traditionally, CD62L imposes lymph node homing on CD62L+ T central memory cells whereas CD62L− T effector memory cells accumulate at peripheral sites (61). Beyond differences in tissue localization, the longevity of T central memory cells is greater than T effector memory cells, which are thought to be more terminally differentiated yet superior at maintaining protection at barrier sites (62). Because sustained proliferation of transitioning effector CD8 T cells maintains low levels of CD62L expression (63) and results in terminal differentiation (64), our data would suggest that the TSLP-driven proliferative burst at the site of infection can drive the formation of T effector memory cells. In support of this theory, blocking the migration of effector CD8 T cells to the lungs abrogated the development of short-lived effector cells (65), perhaps due in part to their inability to access and proliferate in response to TSLP.

In summary, we think that TSLP plays an important role in antiviral immune responses in the lung both by maintaining barrier function and by modulating the proliferation of effector CD8 T cells in situ. With regard to the latter, our data support a model in which TSLP directly regulates respiratory antiviral CD8 T cells by acting as a rheostat to balance protective immunity and limit immunopathology. Under inflammatory conditions, including influenza infection, respiratory epithelial cells temporally increase local TSLP levels (Fig. 1) (24, 36). Consequently, activated CD8 T cells immigrating into the respiratory tract are juxtaposed to TSLP, which supports a limited, additional proliferative burst to these effector cells in situ (Fig. 6B), quantitatively enhancing the peak number of anti-influenza CD8 effector T cells seeding the respiratory tract as well as memory CD8 T cells derived from this pool (Fig. 4B). Because data, including our own (Fig. 5A), suggest that activated and memory CD8 T cells in respiratory mucosal tissue are less responsive to the proliferative affects of γc cytokines, such as IL-7 and IL-15 (11, 12), TSLP may provide the dominant proliferative signal available to these cells early in situ. However, our data suggest that the modest proliferation supported by TSLP may affect the long-term destiny of the respiratory anti-influenza CD8 memory T cells in that the numerical advantage may be at the expense of a shorter lived, more terminally differentiated fate.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. R. Tripp for access to the LSR II. We also thank D. Campbell for helpful discussions regarding the manuscript, as well as M. Field for technical assistance.

Footnotes

  • This work was supported by the University of Georgia and by National Institutes of Health Grant AI081800 (to K.D.K.).

  • Abbreviations used in this article:

    7-AAD
    7-aminoactinomycin D
    BAL
    bronchoalveolar lavage
    γc
    γ-chain
    DC
    dendritic cell
    HA
    hemagglutinin
    i.n.
    intranasal(ly)
    MdLN
    mediastinal lymph node
    MLE
    mouse lung epithelial
    MOI
    multiplicity of infection
    NP
    nuclear protein
    p.i.
    postinfection
    RT-qPCR
    real-time quantitative PCR
    TSLP
    thymic stromal lymphopoietin
    TSLPR
    thymic stromal lymphopoietin receptor
    WT
    wild-type.

  • Received August 6, 2013.
  • Accepted December 28, 2013.

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