Fig 2: Metabolized PYC up-regulates the expression of cluster of differentiation (CD) 83, TLR1, TLR5, and TLR6. THP-1 macrophages were incubated for 18h with dialysates collected after 4 h and 10 h of metabolism or PAM3 as positive control. After 18h of incubation, the cells were harvested and the expression of (A) CD83, (B) TLR1, (C) TLR5, and (D) TLR6 was determined and expressed as differences in mean fluorescence intensity (MFI). PYC samples were metabolized in the presence of E: enzymes; BAC: intestinal bacteria (human faecal suspension). All data are expressed as the mean ± SD of n = 4 independent replicates. p-values < 0.05 are considered statistically significant, as analyzed with one-way ANOVA with Tukey post hoc comparison test (GraphPad Prism). Significant differences are indicated by asterisks: * p < 0.05; ** p < 0.01.

Fig 3: Intra-melanoma DP T cells induce DC maturation in a contact-dependent manner. iDCs were co-cultured for 36 h with intra-melanoma SP CD4+, DP and SP CD8+ CD40L- T-cell subpopulations pre-stimulated for 6 h with OKT3 at ratio of 1:2, and the expression of maturation markers (CD80, CD83, CD86 and HLA-DR) was determined by flow cytometry. (A) Representative flow cytometry analysis of cell surface expression of different maturation markers. DCs were stained with either the isotype control mAb (filled histrogram) or with the indicated maturation marker mAb (open histograms). Results are expressed as the relative fluorescence intensity (RFI). (B) RFI of CD83, CD80, CD86 and HLA-DR expression on DCs after maturation with either SP CD4+ (black diamonds), DP (white circles) and SP CD8+ CD40L- (black triangles) T-cell subpopulations derived from TIL of M125, M265 and M305 melanoma patients (n = 3 independent experiments with three different DC donors). Results are expressed as mean ± SEM. (C) Representative flow cytometry analysis of DCs maturation profile after co-culture of intra-melanoma DP T cells with iDCs respectively seeded in the upper chamber and in the lower chamber of a Transwell plate at a 1:2 ratio for 36 h. DCs were stained with either the isotype control (filled histograms) or with CD80, CD83, HLA-DR or CD86 mAbs (open histograms). Data are expressed as the RFI. Control experiments included the standard co-culture of Mo-DCs with or without DP T cells.