Tc CM and NSTc CM from the same donor’s PBMCs were concentrated using a 50-kDa MWCO Amicon Ultra-0.5 Centrifugal Filter Unit (EMD Millipore). Concentrate and flow-through (filtrate) were collected. Concentrated Tc CM was incubated with CD26 peptidase (0.4 U/ml) (Thermo Fisher Scientific) at 37°C for 2 hours, followed by incubation with 2 mM diprotin A (Sigma) at 37°C for 15 min (to inactivate peptidase activity). Concentrate and filtrate from Tc CM and NSTc CM and CD26-treated concentrated Tc CM were tested for induction of PGE2 in MDP-treated monocytes.

CD3 bead–purified T cells and NS T cells from the same donor’s PBMCs washed five times in serum-free PBS were cultured at 15 × 106 cells/ml in serum-free Expi293 Expression Medium (Thermo Fisher Scientific) at 37°C overnight. Tc CM and NSTc CM were collected and concentrated 18-fold using a 50-kDa MWCO Amicon Ultra-0.5 Centrifugal Filter Unit (EMD Millipore), and protein concentrations of Tc CM or NSTc CM were determined by BCA protein assay kit (Thermo Fisher Scientific).

Concentrated Tc CM and NSTc CM were analyzed by liquid chromatography–tandem MS (LC/MS/MS) using Thermo Fisher Scientific Ultimate LC and Fusion Orbitrap MS. Typical settings for quantitative proteomics at FDA’s Facility for Biotechnology Resources were used in the analysis (55). Proteome Discoverer 1.4 (Thermo Fisher Scientific) was used to match MS/MS spectra to peptides using the Swiss-Prot human database. The parameter settings were previously reported (56).

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