TSLP promotes influenza-specific CD8+ T-cell responses by augmenting local inflammatory dendritic cell function

Abstract

Thymic stromal lymphopoietin (TSLP) is a mucosal tissue-associated cytokine that has been widely studied in the context of T helper type 2 (Th2)-driven inflammatory disorders. Although TSLP is also produced upon viral infection in vitro, the role of TSLP in antiviral immunity is unknown. In this study we report a novel role for TSLP in promoting viral clearance and virus-specific CD8+ T-cell responses during influenza A infection. Comparing the immune responses of wild-type and TSLP receptor (TSLPR)-deficient mice, we show that TSLP was required for the expansion and activation of virus-specific effector CD8+ T cells in the lung, but not the lymph node. The mechanism involved TSLPR signaling on newly recruited CD11b+ inflammatory dendritic cells (DCs) that acted to enhance interleukin-15 production and expression of the costimulatory molecule CD70. Taken together, these data highlight the pleiotropic activities of TSLP and provide evidence for its beneficial role in antiviral immunity.

Figure 1

Thymic stromal lymphopoietin (TSLP) is induced during influenza A infection and augments virus-specific CD8+ T-cell responses. Wild-type (WT) and TSLP receptor (TSLPR)-deficient mice were infected intranasally with 50 PFU PR8 influenza A virus. (a) TSLP expression was determined by real-time PCR at the indicated time points after infection. (b) Viral load from whole lung and trachea was determined by quantitative real-time PCR. Expression of influenza matrix protein was normalized to β-actin expression at the indicated time points after infection. The tissue culture infectious dose 50 (TCID 50) was determined on lung homogenate samples at the indicated time points. Data are pooled from two experiments (n=3–5) and are representative of at least four independent experiments. (c) Recruitment of total CD8+ T cells and (d) influenza-specific NP 366–374, PA, and PB-1 CD8+ T tetramer+ cells in the bronchoalveolar lavage (BAL) was assessed by flow cytometry on day 10 after infection (n=5). Data are representative of at least three independent experiments. (e) BAL cells were restimulated in vitro by bone marrow-derived dendritic cells (BMDCs) pulsed with ultraviolet (UV)-inactivated virus for 6 h before analysis of interferon-γ (IFN-γ) production. Data are representative of two independent experiments (n=5–6). (f) Granzyme B expression in total CD8+ T cells from the BAL was determined by flow cytometry. Solid histogram represents fluorescence minus one control. Data are pooled from two experiments (n=4–6). Granzyme B expression in tetramer-specific CD8+ T cells from the BAL was determined by flow cytometry. Data pooled from two experiments (n=3–6). Error bars represent s.e.m. MFI, mean fluorescence intensity.

Figure 2

Thymic stromal lymphopoietin (TSLP) does not regulate survival of lymphocytes recruited to the site of infection. Wild-type (WT) and TSLP receptor (TSLPR)-deficient mice were infected intranasally with 50 PFU PR8 influenza virus. (a) On day 10 after infection, mice were killed and the indicated cell populations infiltrating the bronchoalveolar lavage (BAL) were analyzed for cell death (7-AAD+, Annexin V+) and apoptosis (7-AAD−, Annexin V+) by flow cytometry. 7-AAD, 7-aminoactinomycin D. (b) Expression of the antiapoptotic molecule Bcl-2 (B-cell lymphoma 2) was assessed in the indicated cell populations by flow cytometry. Data are representative of at least three independent experiments (n=4–5).

Figure 3

Thymic stromal lymphopoietin (TSLP) acts indirectly to augment influenza-specific CD8+ T-cell responses. Lethally irradiated wild-type mice (WT) were reconstituted with bone marrow from WT (Ly5.1) and TSLP receptor (TSLPR)-deficient (Ly5.2) mice in a 1:1 ratio. At 8 weeks after reconstitution, chimeric mice were infected with 50 PFU PR8 and influenza-specific responses in the bronchoalveolar lavage (BAL) were monitored on day 10 after infection. (a) Percentage of CD8+ T cells from the WT or TSLPR-deficient compartment was analyzed by flow cytometry. The percentage of NP 366–374 tetramer+ cells within CD8+ T cells of either the WT or TSLPR-deficient compartment was evaluated. (b) Viability of the indicated cells within the WT or TSLPR-deficient compartment was determined by using Annexin V and 7-aminoactinomycin D (7-AAD). Granzyme B expression in (c) total and (d) NP 366–374 tetramer+ CD8+ T cells within the WT or TSLPR-deficient compartment was measured. MFI, mean fluorescence intensity. (e) Cells from the BAL of infected mice were restimulated with bone marrow-derived dendritic cells (BMDCs) pulsed with influenza virus for 6 h in the presence of monensin and the proportion of interferon-γ (IFN-γ)-producing cells within the WT or the TSLPR-deficient CD8+ T-cell compartment was determined. Data are pooled from two independent experiments (n=3–5).

Figure 4

Thymic stromal lymphopoietin (TSLP) does not affect priming of T-cell responses in the draining lymph node but affects DC function in the lung. Wild-type (WT) and TSLP receptor (TSLPR)-deficient mice were administered 50 μg Ova-FITC (ovalbumin-fluorescein isothiocyanate) together with 50 PFU PR8 intranasally. (a) Migration of airway dendritic cells (DCs) transporting Ova-FITC to the lung draining lymph node was monitored by flow cytometry at the indicated time points after challenge. Data are representative of two independent experiments (n=3). (b) WT and TSLPR-deficient mice were infected intranasally with 50 PFU recombinant PR8-SIINFEKL influenza virus. As indicated, either on day 5 or day 7 after infection, CD11c+ cells were sorted by magnetic bead separation from the draining lymph nodes of infected mice and cultured together with carboxyfluorescein succinimidyl (CFSE)-labeled OT-I transgenic CD8+ T cells for 72 h. Black line represents WT mice and solid histogram represents TSLPR-deficient mice. Division index was determined using FlowJo software and represents the average number of divisions a cell has undergone. WT and TSLPR-deficient mice were infected with 50 PFU PR8 and (c) number of total CD8+ T cells and influenza-specific NP 366–374 tetramer+ CD8+ T cells in the mediastinal lymph node was assessed on day 10 after infection by flow cytometry. Data are representative of at least three independent experiments (n=3–6). (d) Mediastinal lymph node cells were isolated from infected mice day 10 after infection and restimulated in vitro by bone marrow-derived dendritic cells (BMDCs) pulsed with ultraviolet (UV)-inactivated virus for 6 h before analysis of interferon-γ (IFN-γ) production. Data are representative of two independent experiments (n=5–6).

Figure 5

Thymic stromal lymphopoietin (TSLP) regulates interleukin-15 (IL-15) trans-presentation by CD11b+ inflammatory dendritic cells (DCs). (a) Wild-type (WT) and TSLP receptor (TSLPR)-deficient mice were infected intranasally with 50 PFU recombinant PR8-SIINFEKL influenza virus. On day 5 or day 7 after infection, CD11c+ cells were sorted by magnetic bead separation from the lungs of infected mice and cultured together with carboxyfluorescein succinimidyl (CFSE)-labeled OT-I transgenic CD8+ T cells for 72 h. Division index was determined using FlowJo software and represents the average number of divisions a cell has undergone. Black line represents WT mice and solid histogram represents TSLPR-deficient mice. (b) IL-15 expression was determined by quantitative reverse-transcriptase–PCR (RT–PCR). Results were first normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, and subsequently to expression by naive lungs to calculate the ΔΔCt. Data were pooled from two independent experiments (n=3–5). (c) IL-15 and IL-15/IL-15Rα protein levels were determined in lung homogenate from infected mice at indicated time points after infection (n=4–5). (d) Mice were infected with 50 PFU PR8 and on day 9 after infection given either phosphate-buffered saline (PBS) or IL15/IL15R complexes (IL-15c) intranasally. Granzyme B expression by influenza-specific CD8+ T cells in the airways was assessed 12 h later. MFI, mean fluorescence intensity. (e) Lung-recruited inflammatory CD11b+ DCs were quantified by flow cytometry at the indicated time points after infection. (f) Mice were infected with 50 PFU PR8 and CD11b+ DCs were sorted by flow cytometry from pooled lungs (n=3–4) of either WT or TSLPR-deficient mice at indicated time points after infection. The IL-15 mRNA level was measured in these DCs. (g) CD70 expression was determined on the CD11b+ inflammatory DC subset on indicated days after infection. Data are representative of at least two independent experiments (n=3–5).

Figure 6

Thymic stromal lymphopoietin (TSLP) enhances pulmonary influenza-specific CD8+ T-cell responses. (a) TSLP does not affect the initiation of adaptive immune response in the draining lymph node. (b) It acts on CD11b+ inflammatory DCs recruited to the lung and (c) induces the production of interleukin-15 (IL-15) and expression of CD70, which enhances influenza-specific CD8+ T-cell responses.