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MATERIAL AND METHODS

Mice

Female BALB/c mice aged 8-10 weeks from Charles River Laboratory (Margate, UK) and DO11.10 mice with OVA-peptide:323–339-specific, T-cell receptor (TCR) transgenic CD4+ T-cells [1] from The Jackson Laboratory (Bar Harbor, ME) were housed under specific pathogen free conditions, with free access to food and water. Mice were used as sources of OVA-specific CD4+ T-cells and bone marrow derived DCs (BM-DC) under experimental protocols approved by our institutional ethics committee and the Home Office, London, UK.

Virus

Human RSV (A2 strain) from LGC Promochem (Teddington, UK) free of chlamydia or mycoplasma contamination, was plaque-purified and grown in HEp-2 cells (LGC Promochem) cultured in complete RPMI 1640 medium, supplemented with 2% FBS, 2mM L-glutamine, 100U/ml penicillin, 100µg/ml streptomycin all from Life Technologies (Paisley, UK). A transgenic RSV strain expressing green fluorescent protein (GFP-RSV),[2] a kind gift from Dr ME Peeples (Ohio Sate University, Columbus, OH) was cultured in the same way.

Generation of BM-DC

The generation of BM-DC was performed as previous described.[3] Briefly, bone marrow cells were obtained by flushing the femoral bones and cultured in 100mm Petri dishes at 37°C in complete RPMI 1640 medium supplemented with 10% heat inactivated FCS and 20 ng/ml mouse recombinant GM-CSF (Life Technologies). During the culture period of 10-12 days, this medium was replaced every three days. Cells were harvested by vigorous pipetting and PBS washes with Versene 1:5000 (Life Technologies).

Lung epithelial cells (LECs)

LA4-cells, a clonal, murine, lung, alveolar type-II epithelial cell line [4] purchased from the LGC Promochem (Teddington, UK) were grown to confluence in 25cm2 tissue culture flasks in Ham’s F-12 medium (Invitrogen, Paisley, UK) containing 10% FCS, 2 mM L-glutamine, and penicillin-streptomycin (100 U/ml, 100µg/ml).

Primary LECs were generated as described.[5] Briefly, lungs were harvested from naïve BALB/c mice. Before harvest, lungs were gently perfused via the right ventricle with 5-10 ml of PBS to remove blood cells from the pulmonary circulation. The trachea was exposed, a catheter was inserted and the lung was filled with 1.5ml of dispase II solution (5mg/ml). After 5 mins, 0.4ml of 1% low-melt agarose was slowly injected at 45ºC. The lung was immediately covered with ice to allow the agarose to solidify. Then lungs were removed and incubated for 40 mins in 2ml of dispase II at room temperature. After digestion, lung cells were dispersed in a Petri dish in 7ml of DMEM-25mM Hepes supplemented with 50μg/ml of DNAse I and filtered through a nylon screen cell strainer (70µm). Viable cells were counted by trypan blue exclusion. LECs were purified by depletion of contaminating mononuclear cells using anti-CD45, anti-CD32/16, anti-CD31 and anti-CD90 antibody and MACS beads (Miltenyi, Bergisch-Gladbach, Germany). After washing, the isolated primary LECs were re-suspended in complete RPMI 1640 medium in 24 well plates and cultured for three days before they were used in co-culture experiments.

To assess the ability of LECs to inhibit DC-induced T cell proliferation, LA4-cells or primary LECs (5 x 105 /well) were seeded into 24 well plates and incubated in F12 medium (Invitrogen) with 10% FCS. After 24 hours, DC/T-cell co-cultures were added directly to LECs. In some co-cultures, direct contact of LA4-cells with DCs or T-cells was prevented by Transwell chambers (Costar, Fisher Scientific Ltd, Loughborough, UK). In antibody neutralization assays, LA4-cells were incubated prior to and during co-culture with DC/T-cells with 1-40µg/ml of functional grade purified anti-mouse PD-1 antibody (Clone J43) or Armenian Hamster IgG as isotype control (both from eBioscience, San Diego, CA), or with anti-mouse TGF-β (1,2,3) (Clone 1D11) or non-specific rat IgG1 (both from R&D Systems, Abingdon, UK) as isotype control.

In infection experiments, 80%-confluent LA4-cells were inoculated with RSV in a multiplicity of infection (MOI) of 0.1 -10, mock infected with the same amounts of UV-RSV or they remained uninfected. 24 hours after infection, LA4-cells were washed and cultured in fresh complete media and DC/T cell cultures were added. To dissect RSV-effects on the inhibitory capacity of LECs, LA4-cells were pre-treated with TSLP, GM-CSF or IL-6 at a final concentration of 10ng/ml (Invitrogen) or with the following toll-like receptor (TLR) agonists in a final concentration of 10µg/ml: lipo-polysaccheride (LPS) from E coli serotype 055 :B5, Poly I:C (both from Sigma-Aldrich, Dorset, UK), CpG-ODN1826 (5'-TCCATGACGTTCCTGACGTT-3') or the control ODN1982 from Life Technologies.

T-cell proliferation assays

DC-induced T cell proliferation was assessed as described.[6] Briefly, following depletion of CD11c+ cells using anti-CD11c coated MACS beads, splenic CD4+ T-cells were isolated from DO11.10 mice by Auto-MACS using anti-CD4 coated MACS beads (all from Miltenyi). Purified CD4+ DO11.10 T-cells were washed, re-suspended at 1x107 cells/ml in PBS and 5µM of carboxy fluoroscein succinimidyl ester(CFSE) (Life Technologies) was added. After 15 minutes incubation at 37°C, unbound dye was quenched by addition of an equal volume of FCS and the cells were washed three times with ice-cold medium containing 10% FCS. BM-DC (1 x 105/well), pulsed overnight with 100µM OVA or with PBS as a control, were incubated with CFSE labelled CD4+ DO11.10 T-cells (5 x 105/well). This co-culture was layered onto LECs or medium as control. After four days non-adherent cells (DC, T-cells) were harvested and reduction in CFSE fluorescence intensity, indicating T-cell proliferation, was determined by flow cytometry. In some experiments, proliferation of T-cells from BALB/c mice was induced by anti-CD3 antibody (10 µg/ml, BD.145-2C11) and irradiated (3000 rad), BM-DC.

Flow cytometry

To avoid non-specific antibody binding, FC-receptor blockade with anti-mouse CD16/CD32 mAb (24G2; BD Biosciences, San Diego, CA) was used prior to staining with the following anti mouse antibodies: anti-DO 11.10 TCR-TRI colour and CD62L-PE (MEL14) from Caltag Laboratories (Burlingame, CA), anti Foxp3 (FJK-16s), GITR-PE (DTA-1), anti-PDL-1-PE (MIH5), and anti-PDL-2-PE (MIH18) from eBioscience, anti-CD4-PE (RM 4-5), anti-CD25-APC (PC61), anti-CD3-PE-Cy5 (17A2) and rat IgG2a (R35-95) and rat IgG2b (A95-1) as isotype controls all from BD Biosciences. Samples were stained at 4°C in PBS with 1% FBS, acquired using a LSR flow cytometer and CellQuest software (both from BD Biosciences) and analysed using WinList software (Verity Software House, Topsham, ME).

ELISA

After cell cultures, supernatants were collected, centrifuged and stored at -80°C until cytokine concentrations were assessed by ELISA using the following ELISA kits: mouse GM-CSF and TSLP (R&D Systems Europe, Abingdon, UK), mouse IL-6 and TNF-alpha (Biosource International, Camarillo, CA); and antibody pairs: mouse IL-1 (capture mAb ALF-162, detection mAb C1150-27), IL-4 (capture mAb 11B11, detection mAb BVD6-24G2), IL-5 (capture mAb TRFK5, detection mAb TRFK4), IL-10 (capture mAb JES5-2A5, detection mAb SXC-1), IL-12p70 (capture mAb 9A5, detection mAb C17.8), IL-17 (capture mAb TC11-18H10.1, detection mAb TC11-8H4.1), IFN-γ (capture mAb A6-18, detection mAb XMG1.2) with protein standards from BD Biosciences. Total TGF- β protein levels were measured by ELISA (R&D Systems). ELISA assays were performed according to manufacturer’s instructions. OD values at 450 nm were measured using an MRXII Dynex spectrophotometer with Revelation F3.21 software (ThermoBioAnalysis SA, France).

RESULTS AND DISCUSSION

Early and prolonged presence of LECs in DC/T-cell co-cultures is required for T-cell inhibition.

In light of the observation that LEC are capable of inhibiting antigen-specific T-cell proliferation, we asked whether this inhibition is dependent on the timing and duration of LEC presence in DC/T-cell co-cultures. DCs and T-cells were transferred at various time points after the start of co-culture onto LA4 cell layers where they remained until T-cell proliferation was assessed on day four (OL fig 1a). Only addition of LA4-cells in the first 24 hours of DC/T-cell co-cultures resulted in T-cell inhibition, while T-cells proliferated normally if LA4-cells were added at 48 hours of culture or later. This failure of LECs to inhibit T-cell proliferation suggests that the T-cells were fully activated after 24 hours of co-culture with DCs and that LECs were not able to reverse T-cell activation and subsequent proliferation. Thus, presence of LECs prior to T-cell activation is required for T-cell inhibition.

In separate experiments (OL fig 1b), we asked if the duration of LEC presence in DC/T-cell co-cultures determines the degree of T-cell inhibition. DC/T-cell co-cultures were started in the presence of LA4-cells, were subsequently removed from these LECs different time points and continued until day 4. The short presence of LA4-cells in DC/T-cell co-cultures for only 8 hours significantly reduced T-cell proliferation. The degree of T-cell inhibition increased with the time that LA4-cells were present in DC/T-cell co-cultures with almost complete inhibition after 72 hours. These observations indicate that prolonged interaction between LECs and DC/T-cells was required to inactivate the majority of T-cells. However, even a short interaction with LECs is sufficient to induce some degree of T-cell inhibition and this is not fully reversible after removal of DC/T-cells from the LEC environment.

Infection of LA4-cells by GFP-RSV

To ascertain that LA4-cells can be infected with RSV we used a genetically modified strain which expresses GFP (GFP-RSV). After 24 hours LA4-cells infected with live GFP-RSV, but not cells infected with UV-inactivated virus or non-infected LA4-cells, displayed robust green fluorescence indicating infection and replication of GFP-RSV in these cells (OL fig 2). This result was confirmed by quantitative PCR of the RSV-L gene, increased copy numbers of which were only detected in LA4-cells infected with live, but not with UV-inactivated RSV (data not shown).

Pro-inflammatory cytokines, secreted by LECs upon RSV-infection, do not reduce their T-cell inhibitory capacity.

We infected LA4-cells with RSV or UV-inactivated virus as a control and assessed the production of TSLP, GM-CSF and IL-6. TSLP is known to promote allergic airway inflammation in asthma,[7] IL-6 can override inhibitory effects of Tregs [8] and GM-CSF is the main growth and differentiation factor for DCs.[9] In sham-infected cultures, TSLP and IL-6 were hardly detectable and GM-CSF was secreted at low levels. Following RSV infection, all of these pro-inflammatory cytokines were secreted in substantial amounts (OL fig 3a). LA4-cells did not secrete any of the following cytokines: IFN-(, IL-4, IL-5, IL-10, IL-12, IL-17 or TNF-α. We next incubated LA4-cells with TSLP, GM-CSF, IL-6, supernatant from RSV-infected LA4-cells, or PBS as a control and added DC/T-cells to these LECs after 24 hours. None of these potent pro-inflammatory cytokines nor the total supernatant from RSV-infected LA4-cells reduced the ability of LECs to inhibit T-cell proliferation, indicating that they are not the factors mediating this effect of LECs.

FIGURE LEGENDS

OL 1: Inhibition of antigen-specific and non-specific T-cell proliferation by primary LECs

(A) CFSE-stained DO11.10 T-cells were cultured alone or co-cultured with OVA-pulsed BM-DC (ratio 10:1) in the presence or absence of fully confluent primary LECs monolayers. (B) CFSE-stained spleen CD4+ T-cells from BALB/c mice were cultured alone or co-cultured with Dynabeads mouse CD3/CD28 T-cell expander (Invitrogen) in the presence or absence of fully confluent primary LECs monolayers. (C) CFSE-stained spleen CD4+ T-cells from BALB/c mice were cultured alone or co-cultured with normal BM-DC (ratio 10:1) plus 10µg/ml anti-CD3 antibody (Clone 145-2C11, from BD Biosciences) in the presence or absence of fully confluent primary LECs monolayers. DC-induced T-cell proliferation was assessed by flow cytometry measuring CFSE dilution. The dot plots shown are representative of three independent experiments. The figures in the dot plots indicate the percentage of non-proliferated T-cells.

OL Figure 2: Timing and duration of LA4-cell presence determine the degree of T-cell inhibition.

(A) In order to determine when during DC/T-cell culture the presence of LECs is required for T-cell inhibition, DC/T-cell co-cultures were transferred onto LA4-cells after 8, 24, 48 or 72 hours of co-culture and T-cell proliferation was assessed after 4 days of total DC/T-cell culture time. (B) To determine the duration of co-culture with LECs required for T-cell inhibition, DCs and T-cell were removed from the LA4 cell layers after 8, 24, 48 or 72 hours and the DC/Tcell co-culture was continued in the absence of LECs for a total culture time of 4 days when T-cell proliferation was assessed. The graphs show means ±SEM of the percentage of non-proliferated T-cells from a representative experiment (6 samples per group) of three independent experiments. Significant differences versus DC/T-cells without LA4-cells: *p ................
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