Mixed lymphocyte reaction assay

The mixed leukocyte reaction (MLR) assay was performed as described previously (Rudolf and others 2001; Fausch and others 2002; Muul and others 2008). Briefly, HLA-A*0201 LCs were treated (or not) with HPV VLPs and/or IRX-2 and cocultured with untreated allogeneic HLA-mismatched (non-HLA-A2) CD4⁺ and CD8⁺ T cells isolated from different donor PBMCs by negative magnetic separation (Miltenyi Biotec, San Diego, CA). Responder (R) T cells and irradiated stimulator (S) LCs were cultured at an R:S ratio of 20:1 in a 96-well round bottom plate in replicates of 6 per treatment for 5 days. T cells and LCs, each cultured alone, and T cells cultured with autologous PBMCs served as negative controls, while PHA-treated T cells served as a positive control. Five days later, ³H-thymidine was added, and after an additional 18 h, radioactive ³H-thymidine-pulsed cells were harvested and radioactivity counted on a TopCount microplate liquid scintillation counter to quantify cell proliferation (PerkinElmer, Waltham, MA). In separate experiments, purified T cells were cultured with IRX-2 alone to determine the direct effect of the reagent on T-cell proliferation.