Figure 2
From: Exhaustion of CD4+ T-cells mediated by the Kynurenine Pathway in Melanoma
Inhibition of CD4+CD25− T-cell proliferation by MCLs associated with KP enzymatic alteration. (a) Schematic workflow of the experimental design (b) Measurement of CD4+ T-cell proliferation in vitro by CFSE dilution alone and in culture with MCLs (c,d) IFNγ secretion and IFNγ expression levels of CD4+ T cells in culture with MCLs by ELISA and flow cytometry (e) Measurement of CD4+ T-cell proliferation in vitro by CFSE dilution with medium (RPM1640) and with conditioned medium derived from CD4+ and MCLs co-culture (f–h) FOXP3, PD1 and CTLA4 protein expression from educated CD4+ T-cells by flow cytometry (i), IDO1 protein expression from educated MCLs determined by flow cytometry (j) Gating strategies of IDO1 positive MCLs cultured with CD4+ T-cells (k,l) KYN and KYNA production by LC-MS/MS. Graphs show individual data, and horizontal lines show mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.005 by independent samples t-test, n > three biological replicates cultured with four different melanoma cell lines.
