The following antibodies were used: Alexa Fluor 700–conjugated CD4 (RPA-TA; BD), allophycocyanin (APC)-Cy7–conjugated CD25 (M-A251; BD), PE-Cy5­–conjugated CD127 (eBioRDR5; eBioscience), Pacific blue–conjugated CD14 (M5E2; BD), FITC-conjugated Ki67 (35 Ki-67; BD), PE-conjugated RORγτ (AFKJS-9; eBioscience), Alexa Fluor 647–conjugated FoxP3 (PCH101; eBioscience), PE-conjugated FoxP3 (236A/E; eBioscience), FITC-conjugated membrane TNF (FAB210F; R&D Systems), PE-conjugated TNF-RII (22235; R&D Systems), PE-conjugated STAT5 (PY694; BD), FITC-conjugated active Caspase 3 (C92-605; BD), FITC-conjugated IL-2 (MQ1-17H12; eBioscience), Alexa Fluor 647–conjugated Il-17A (BL168; BioLegend), PE-Cy7–conjugated IFN-γ (B27; BD), soluble anti-CD3 (HIT-3a; eBioscience), anti-CD28 (CD28.2; eBioscience), anti–TNF-RII antagonist (22221; R&D Systems), anti–TNF-RII agonist (MR2-1; Hycult Biotech), and anti–IL-2 neutralizing mAb (MQ1-17H12; BD). STAT5 inhibitor N′([4-oxo-4H-chromen-3-yl]methylene) nicotinohydrazide (EMD Millipore) was also used. Of relevance, FITC-conjugated membrane TNF (FAB210F; R&D Systems) does not interfere with membrane TNF’s binding to TNF-RII (Gerspach et al., 2000 (link)).
Nguyen D.X, & Ehrenstein M.R. (2016). Anti-TNF drives regulatory T cell expansion by paradoxically promoting membrane TNF–TNF-RII binding in rheumatoid arthritis. The Journal of Experimental Medicine, 213(7), 1241-1253.
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