CD81 negatively regulates heterotypic fusion of macrophages with infected Jurkat T cells through the RhoA/ROCK/actomyosin pathway. (A–D) Role of CD81 in infection of macrophages by fusion with infected CD4⁺ T cells. (A) Quantification by flow cytometry of infection of MDMs incubated with isotype control (Control) or antibody targeting CD81 (anti-CD81) before co-culture with infected Jurkat cells, normalized to the isotype control condition (n = 6 donors, mean ± SD). (B) MDMs were transfected with non-targeting siRNA (siCTRL) or targeting CD81 (siCD81) and co-cultured during 24 h with infected Jurkat cells. Quantification by flow cytometry of MDM infection, normalized to the siCTRL condition (n = 8 donors, mean ± SD). (C) Same experiment as in A except that Jurkat cells were replaced by autologous primary T cells (n = 7 donors, mean ± SD). (D) Same experiment as in A except that MDMs were replaced by macrophages isolated from broncho-alveolar lavages (n = 3 donors, median ± interquartile range). (E and F) Involvement of CD81 in the regulation of myosin II activity. (E) Quantification of MLC activation. Left: Representative images of Western blot analysis illustrating the phosphorylation of MLC (p-MLC, top) and expression of total myosin IIA (middle), with actin as loading control (bottom). Right: Quantification of p-MLC/actin ratio, normalized to the siCTRL condition (n = 9 donors, median ± interquartile range). (F) Quantification of RhoA activation: After CD81 silencing, MDMs were lysed and then pull-down of active GTP-Rho was performed using GST-RBD beads. Left: Representative images of Western blot analysis illustrating the expression of active RhoA (GTP-RhoA, top) and total RhoA (middle), both revealed with a RhoA antibody with actin as loading control (bottom). Right: Quantification of GTP-RhoA/total RhoA, normalized to actin; siCTRL condition serves as a reference (n = 6 donors, median ± interquartile range). Statistical analyses: (A–C) Paired t test and (D–F) Wilcoxon test. * P ≤ 0.05; ** P ≤ 0.01.