Pbmcs

Human peripheral blood mononuclear cells (PBMCs) were purchased from Lonza. Healthy human salivary gland epithelial cells (SGECs) were isolated and cultured as we reported previously [24 (link)]. The SGEC-PBMC coculture experiment was based on a published protocol [25 (link)]. Briefly, SGECs were seeded at 1?×?10⁵ cells per well into 12-well plates and cultured in Keratinocyte serum-free medium (SFM, Life technology) with poly I:C (5?μg/ml, InvivoGen, tlrl-picw) for 12 hours to allow attachment, stimulation of autoantigen synthesis, and IL7 expression essential for SS progression. After removing the SFM and washing with PBS, 2?×?10⁴ PBMCs per well were added in LGM-3 lymphocyte growth medium (Lonza) containing 10% FBS and phytohemagglutinin-P (PHA-P, 5?μg/ml, Sigma-Aldrich, L8754) to activate T cells. After 4 days of coculture, SGECs and PBMCs were harvested together and analyzed for gene expression by qRT-PCR.

Cryopreserved human peripheral blood mononuclear cells (PBMCs, Lonza, Walkersville, MD, USA) were resuspended in RPMI-1640 medium containing 10% FBS. The PBMCs were cultured in a 0.4 mm trans-well insert for a 6-well plate at a concentration of 3 × 10⁶ cells per well in a 2 mL culture medium. The cells were incubated for 2 days in the presence of 1 and 2.5 μg/mL of the SEA or the medium alone (unstimulated). The insert was then transferred onto the top of the LX-2 cells (in a 6-well plate, 2 × 10⁵ cells per well) for 2 days. The culture supernatants were collected and stored at −80 °C for TGF-β cytokine measurement. The LX-2 cells were then washed and harvested for real-time PCR analysis.