Figure 4

SLP-76-ADAP regulates the VS formation between T-cells and HIV-1-pulsed DCs. Jurkat, JDAP or J14 cells were incubated for 30 min with mature human DCs that were pre-pulsed with HIV1-gag-GFP VLPs. Conjugate formation and the presence of HIV1-gag-GFP at the VS interface was assessed. (A) Example of a conjugate shows that anti-ADAP staining in T-cells and HIV1-gag-GFP VLPs in DCs were peaked at the interface between DC and T cell. (B) Jurkat cells stably overexpressing GFP, ADAP/GFP or M12/GFP were incubated for 30 min with HIV1-gag-GFP-pulsed human DCs. Represent examples are shown in right panels. Histograms show the percent of conjugate formation (left panel; P = 0.0198 left, P = 0.0068 right) and colocalization of HIV1-gag-GFP and LFA-1 at the VS (right panel; P = 0.0107 left, P = 0.0022 right). (C) Deficiency of ADAP (JDAP) or deficiency of SLP-76 (J14) impaired HIV1-gag-GFP and LFA-1 localization at the interface between Jurkat cells and DCs. Histograms show the percent of conjugate formation (upper panel, P = 0.0011 left, P = 0.043 right) or colocalization of HIV1-gag-GFP with LFA-1 at the VS (lower panel, P = 0.0006). (D) Human primary CD4⁺ T cells were transfected with the scramble siRNA or siRNA ADAP for 48 Hrs, which were then incubated for 30 min with mature human DCs pre-pulsed with HIV1-gag-GFP VLPs. The conjugate formation (P = 0.0241) and HIV1-gag-GFP at the VS interface (P = 0.0018) were analyzed and showed in histograms. Typical examples were shown in right panel. Typical examples are shown in right panel (* represents p = <0.05, ** represents p = <0.01, *** represents p = <0.001).