PDI inhibitors significantly decrease fusion between CD4⁺/CXCR4⁺ and Env⁺ cells. CD4⁺/CXCR4⁺ Sup T-1 (target) cells were treated with PDI inhibitors DTNB or bacitracin (A), as well as anti-PDI MAb or anti-CD3 control MAb (B) (30 minutes, 37°C), then washed and added to Env-expressing cells to assess fusion. (C) For reciprocal experiments to determine the effect of PDI inhibitors on the Env⁺ cells, CHO-160 cells were incubated under the conditions noted earlier in the presence and absence of DTNB (1 or 5 mM). Cells were then washed and cocultured with untreated Sup T-1 cells. Fusion of treated Sup T-1 and untreated CHO-160 cells was compared with fusion of untreated Sup T-1 and treated CHO-160 cells. Fusion was measured by fluorescent dye (Calcein am) redistribution from labeled Sup T-1 to unlabeled Env⁺ cells. Fusion was not observed in the control experiments when CD4⁺/CXCR4⁺ cells were mixed with Env^– cells or in the presence of T-20/T-21 peptides, which are potent inhibitors of HIV-1 Env-mediated fusion³  (data not shown). Fusion (%) represents the ratio of bound, dye-redistributed Sup T-1 cells to the total number of bound Sup T-1. Each point (A) or bar (B) represents mean fusion ± SE for 200 cells counted.