Adalimumab promoted the interaction between monocyte membrane TNF and TNF-RII expressed by T reg cells. (A) Purified T reg cells and monocytes from RA patients were treated with biotinylated adalimumab or etanercept for 30 min. Representative imaging flow cytometry (ImageStream) and cumulative data of T reg cell (Treg)–monocyte conjugate formation in the presence of either anti-TNF agent. (B) Representative ImageStream plots and cumulative data reveal the percentage of conjugates coexpressing either anti-TNF agent or TNF-RII. (C) Cumulative data depicting membrane TNF (mTNF) and either adalimumab or etanercept intensity in the valley mask of the conjugate synapse. (A–C) n = 5. Data were obtained from three experimental repeats. **, P < 0.01; ***, P < 0.001 using Kruskal-Wallis tests with Dunn’s post-hoc analysis. Error bars represent mean ± SE. (D) Representative merged images of CD4⁺ T reg cell, CD14⁺ monocyte, membrane TNF, TNF-RII, and anti-TNF. The BDS index (colocalization) of membrane TNF and TNF-RII at the conjugate interface in monocyte–T reg cell cultures treated with or without adalimumab is shown. Data are representative of three experimental repeats.