Proximity ligation assay shows the proximity of CD2-CD58 and CD4-HLA-II in vitro and in HL tissue. (A) Results of PLAs of L428 WT, CD58-KO, CD54-KO, CIITA-KO, the CD58⁻ KMH2 or the HLA-II⁻ DEV cell line cocultured with matched PBMCs for CD2-CD58 and CD4-HLA-II (red signal). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue signal). Quantification of the number of red dots at the tumor cell–T-cell interface of CD2-CD58 (B) and CD4-HLA-II (C) for multiple rosettes using 1 HLA-II–matched donor. Each point in the graph indicates the average number of red dots per attached cell in a single rosette. (D) Immunohistochemical staining for CD2 and TCR in snap-frozen primary HL tissue. Arrows indicate tumor cells. (E) PLA staining (red signal) for CD2-CD58 and CD4-HLA-II on 2 different tissue samples from patients with primary HL. Nuclei were counterstained with DAPI (blue signal). Asterisks indicate tumor cells. In panels A and E, PLA signals were visualized with a Leica DM4000B microscope equipped with a Leica DFC345FX Camera and LAS V4.8 software. All images are representative; original magnification ×400. Red dots indicate the proximity (within 40 nm) of the 2 proteins studied. Statistical significance was calculated with a 1-tailed Mann-Whitney U test, with all conditions compared with the WT. *P < .05; ****P < .0001.