Figure 5. BRAF(V600E) can induce T-cell suppression through IL-1 mediated upregulation of PD-1 ligands and COX-2 on TAFs.

(A) Flow cytometric analysis of surface PD-L1 expression on TAFs cultured overnight with IL-1α, IL-1β, IL-6, TNF-α, or conditioned medium from cultured primary melanocytes transduced with lentiviral vectors expressing BRAF(wt)-GFP, BRAF(V600E)-GFP, or GFP alone. As indicated, conditioned media experiments were also performed in the presence of isotype control or anti-IL1α and anti-IL1β blocking antibodies. (B) Interferon-gamma release by T2-stimulated MART-1 reactive TIL in the presence of melanoma patient-derived TAFs previously exposed to conditioned media from BRAF(V600E) mutant-expressing melanoma cell lines that were either untreated or treated with the BRAF(V600E) inhibitor vemurafenib. Results from five different TAF lines are shown. (C) To assess the relative contributions of IL-1α/β, COX-2, and PD-1 ligands in the induction of T-cell suppression, three melanoma TAF lines were pre-treated with conditioned media from untreated or vemurafenib-treated melanoma cell lines (WM793p2 and EB16-MEL), in the presence of either IL-1α/β blocking antibodies or the COX-2 inhibitor NS398. Pre-conditioned TAFs were then incubated with MART-1-reactive TIL and MART-1 peptide-pulsed T2 cells in the presence of isotype control antibody or antibodies specific for PD-L1 and PD-L2. Asterisks indicate statistical significance (P < 0.05); ns, not significant.