Tgf β

PBMC were depleted of CD8+ T cells by MACS using anti-CD8+ direct beads (Miltenyi Biotec), according to manufacturer’s instructions and separated on an AutoMACS Pro. The resulting CD4+ T cell & Antigen Presenting Cell (APC) PBMC were resuspended in either X-VIVO 15, X-VIVO 15 with 4ng/ml TGF-β and 10ng/ml IL-10 (both Miltenyi Biotec), neat Mock infected monocyte secretome, neat UV irradiated infected monocyte secretome or neat Latent Infected Monocyte secretome. The cells were then plated in 48-well tissue culture plates and incubated overnight at 37°C in a humidified CO₂ atmosphere. After 24 hours incubation, the cells were stimulated with 1µg/ml anti-CD3 and 0.5µg/ml anti-CD28 (both Mabtech AB) and overlapping peptide pools for HCMV proteins (43 (link)) resulting in a 1:2 dilution of the secretomes and TGF-β/IL-10 mix. Following a further 24-hour incubation at 37°C in a humidified CO₂ atmosphere, the plates were centrifuged, and supernatants harvested and then analyzed for the production of IFN-γ by ELISA. Full details of proliferation assays used to measure whether latent secretomes can suppress CD4+ T cells can be found in the supplementary material (Section 1.8).

BAL concentrations of lactoferrin (Assaypro LLC, St. Charles, MO, USA), SLPI (FineTest, Wuhan, China), lysozyme (Assaypro), LL-37 (HycultBiotech, Plymouth Meeting, PA, USA), IL-10 (Immunotools), TGF-β (Mabtech, Nacka Strand, Sweden), TNF-α (Mabtech), and IL-17A (PeproTech EC, London, UK) from ILD patients were determined using specific ELISA kits according to the manufacturers’ instructions and using the specific standard curves of recombinant molecules. The limits of detection were as follows: 0.313–20 ng/mL for SLPI, 62.5–4000 pg/mL for lactoferrin, 0.078–5 ng/mL for lysozyme, 0.14–100 ng/mL for LL-37, 15.62–1000 pg/mL for IL-10, 62.5–4000 pg/mL for TGF-β, 15.62–1000 pg/mL for TNF-α, and 15.62–1000 pg/mL for IL-17A.