PV-267 inhibits MBP Ag specific pro-inflammatory cytokine production and proliferation of BC3 T cells. MBP specific BC3 T cells were co-cultured with PV-267 pre-treated autologous APCs and MBP87-99 peptide for human IFN-γ ELISPOT assay as described in Materials and Methods. Shown are representative results of (A) quantified spots at each PV-267 titration, (B) percentage of inhibited response from at least 3 independent experiments, and (C) effect of PV-267 on BC3 T cells in response to anti-CD3 mAb stimulation as control (MEAN±SD). After MBP specific BC3 T cells were co-cultured with PV-267 pretreated autologous APC and medium, Ag or with positive control (PTX/PHA) for 24 h; supernatants were collected and analyzed using Bio-Plex assay for human cytokines. Shown are pooled results of proinflammatory cytokine production of (D) TNF-α, (E) IL-1ra, (F) IL-1β, (G) IL-6, (H) IL-17 and (I) MIP-1a from 3 independent experiments (mean ± SD). (J) MBP specific BC3 T cells were labeled with CFSE then co-cultured with PV-267 pretreated autologous APC and MBP87-99 or mitogen for 5 days followed by flow cytometry analysis as described in Materials and Methods. Shown are pooled results (top) of percentage of proliferated BC3 T cells in response to MBP87-99, anti-CD3 mAb and PHA (MEAN±SD), as well as representative flow cytometry histograms (bottom) from 3 - 4 independent experiments. * indicates significant difference between 0 μg/ml PV-267 and 200 μg/ml PV267 treated groups (p < 0.05, t-test).