Anti il 15rα

Draining lymph nodes (DLN) and conjunctivae were collected from DED mice and cultured in RPMI (Invitrogen) supplemented with 10% FBS. Alternatively, single cell suspensions were prepared from DLN and CD4⁺ T cells were enriched using the negative isolation kit (Miltenyi Biotec). Subsequently, the CD44^hiCD62L⁻ subpopulations were further sorted using MoFlo FACS sorter (Dako Cytomation). The tissue explants or CD44^hiCD62L⁻CD4⁺ cells were treated with anti-IL-7 (10 μg/ml, R&D Systems), anti-IL-15 (5 μg/ml, eBioscience), anti-IL-7 (10 μg/ml) + anti-IL-15 (5 μg/ml), anti-IL-7Rα (10 μg/ml, R&D Systems) + anti-IL-15Rα (10 μg/ml, R&D Systems) Abs, IL-7 (20 ng/ml, PeproTech), IL-15 (20 ng/ml, PeproTech), or IL-7 (20 ng/ml) + IL-15 (20 ng/ml) for 72 hours. Memory Th17 cells were then examined by flow cytometry.

STAT5 signaling: PBMCs (1 × 10⁶ cells /mL) were stimulated overnight with IL15 (7.5 ng/mL; CellGenix)(21 (link)), harvested, then serum-starved for 1hr in the presence of TG101348, ruxolitinib, or DMSO control. The cells were then pulsed with IL2 (EC₅₀ 50 IU/mL; Chiron), IL15 (EC₅₀ 2.7 ng/mL; CellGenix), allogeneic moDCs or LCs (1:1 ratio) for 15 minutes followed by fixation (CytoFix; BD Biosciences)and permeabilization. Cells were stained for CD3 (Clone UCHT1) and CD56 to identify NK cells (CD3⁻CD56⁺) and intracellular Alexa Fluor 647 anti-STAT5 (pY694; BD Biosciences). Where indicated, IL15Rα was blocked on both moDCs and LCs with anti-IL15Rα(5μg/mL)or IgG control (R&D Systems).
STAT4 signaling: PBMCs (1 × 10⁶ cells /mL) were stimulated for 2 d with 100 IU/mL IL2 (Chiron) to facilitate optimal IL12R induction(22 (link)). Cells were harvested and serum-starved for 1 hour in the presence of TG101348, ruxolitinib, or DMSO. Cells were then pulsed with allogeneic moDCs or LCs (1:1 ratio), matured moDC or LC supernatant, or 100 IU/mL IL12p70 (R&D Systems) for 90 min (23 (link)). pSTAT4 expression was ascertained using an intracellular PE- or Alexa Fluor 647-conjugated anti-STAT4 (pY693; BD Biosceinces).
STAT3 signaling: This was assessed in JAK inhibitor-treated T cells as published(3 (link)).