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. 2009 Apr 1;83(12):6171–6183. doi: 10.1128/JVI.02163-08

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FIG. 7. — Role of the VS during HSV cell-cell spread to T cells. (A) A CD8⁺ T-cell clone was preincubated for 20 min at 37°C with either anti-LFA-1 blocking antibody (CD11a) or anti-CD28 antibody at the indicated concentrations before addition to uninfected fibroblasts (no virus) or fibroblasts infected with HSV-1(F) at an MOI of 10 for 16 h at 37°C. After 2 h at 37°C, the T cells were removed, washed with citrate buffer, further incubated at 37°C for 16 h, stained for CD3 and HSV antigens, and then analyzed by flow cytometry. (B) A CD8⁺ T-cell clone was coincubated at 37°C with mock-infected fibroblasts or fibroblasts infected with HSV-1(F) at an MOI of 10 for 16 h at 37°C in the absence or presence of neutralizing anti-gD antibody at the indicated dilutions. After 2 h, the T cells were removed, washed with citrate buffer, further incubated at 37°C for 16 h, stained for CD3 and HSV antigens, and then analyzed by flow cytometry. (C) PBMC were incubated for 20 min at 37°C with either culture medium (untreated; no Tx), culture medium with PMA (50 ng/ml), or culture medium with PMA and anti-LFA-1 blocking antibody (PMA+anti-LFA-1), and then added for 2 h to fibroblasts infected with HSV-1(F) at an MOI of 10 for 16 h. In one group the HSV-infected fibroblasts were preincubated with neutralizing anti-gD antibody (1:50 dilution) for 20 min prior to the addition of PMA-stimulated PBMC (PMA+anti-gD). After the 2-h coincubation, the T cells were removed, washed with citrate buffer and PBS, further incubated at 37°C in culture medium for 24 h, stained for CD3 and HSV antigens, and analyzed by flow cytometry. (D) Jurkat T cells, unstimulated PBMC, and PHA-activated PBMC (5 μg/ml 2 days at 37°C) were exposed for 2 h at 37°C to either fibroblasts that were infected with HSV-1(F) at an MOI of 10 for 16 h at 37°C and washed with PBS prior to the addition of T cells (Cell) or the medium (16 to 18 h postinfection) collected from fibroblasts that were infected with HSV-1(F) at an MOI of 10 for 16 h, washed with PBS, and incubated in medium for 2 h (16 to 18 h postinfection) (Supernatant). In both cases after a 2-h incubation, T cells were removed, spun, washed with citrate buffer and PBS, further incubated at 37°C for 24 h, stained for CD3 and HSV antigens, and analyzed by flow cytometry. (E) PBMC were resuspended for 30 min in either HEPES buffer, HEPES buffer plus Mg2⁺-EGTA, HEPES buffer plus PMA-Ca2⁺, or HEPES buffer plus PMA and then incubated for 1 h at 37°C with fibroblasts infected with HSV-1(F) at an MOI of 10 for 16 h. At the end of the coincubation, the PBMC were removed, washed with citrate buffer and PBS, further incubated in culture medium for 24 h, stained for CD3 and HSV antigens, and analyzed by flow cytometry. (F) PHA-activated PBMC (5 μg/ml 2 days at 37°C) were added for 2 h at 37°C to either uninfected fibroblasts (24 h; Mock) or HSV-infected fibroblasts (MOI of 10) at different times postinfection in the absence (no Ab) or presence of anti-LFA-1 blocking antibody (+LFA-1); PBMC were then removed, washed with citrate buffer and PBS, further incubated at 37°C for 20 to 25 h, stained for CD3 and HSV antigens, and then analyzed by flow cytometry. hpi, hours postinfection.