Figure 4
From: Exhaustion of CD4+ T-cells mediated by the Kynurenine Pathway in Melanoma
The district CD4+ T-cell behaviour in co-culture set-up links to KYNA production. (a) The proliferation of CD4+ T-cells in the presence of V600E and V600 wt MLCs (b,c) KYN and KYNA production by HPLC after 48 h in co-culture set-up (d) AhR mRNA expression in V600E and V600 wt MLCs (e) CD4+ T-cells in co-culture set-up (f) PD-L1 mRNA expression in V600E and V600 wt MLCs in after 5 days co-culture set-up were analysed by real-time PCR and normalized to RPL13A in CD4+ T-cells (g) show representative images of gating strategy (left panel), IFNγ production (right upper panel) and CD4+ T-cell proliferation (right lower panel) in different concentrations of KYNA (h) Cell toxic concentration of KYNA was defined using viability assay by flow cytometry (i,j) CD4+ T-cell proliferation and IFNγ production upon KYNA treatment in five different concentration (0,005–50 µM) (k) BRAF V600E cell lines transiently transfected with expression plasmids encoding BRAF wt and corresponding lysates were subjected to immunoblotting with the indicated antibodies (l,m) the proliferation of CD4+ T-cells (three different donors) and IFNγ production in culture with transfected cell lines (SK.mel-28 and DFB) on day five by flowcytometery. Graphs show individual data, and horizontal lines show mean ± s.e.m. ****P ≤ 0.0001, ***P ≤ 0.0005 **P ≤ 0.001, *P ≤ 0.05, the Tukey multiple comparison procedure was used to determine significance and adjusted P values for the differences in group means can be seen in Table S6, n > three biological replicates cultured with four different melanoma cell lines.
