Abstract
Immune checkpoint inhibitors that target the programmed cell death protein 1 (PD1) pathway have revolutionized the treatment of patients with advanced metastatic melanoma. PD1 inhibitors reinvigorate exhausted tumor-reactive T cells, thus restoring anti-tumor immunity. Tumor necrosis factor alpha (TNFα) is abundantly expressed as a consequence of T cell activation and can have pleiotropic effects on melanoma response and resistance to PD1 inhibitors. In this study, we examined the influence of TNFα on markers of melanoma dedifferentiation, antigen presentation and immune inhibition in a panel of 40 melanoma cell lines. We report that TNFα signaling is retained in all melanomas but the downstream impact of TNFα was dependent on the differentiation status of melanoma cells. We show that TNFα is a poor inducer of antigen presentation molecules HLA-ABC and HLA-DR but readily induces the PD-L2 immune checkpoint in melanoma cells. Our results suggest that TNFα promotes dynamic changes in melanoma cells that may favor immunotherapy resistance.
Highlights
Pathway have revolutionized the treatment of patients with advanced metastatic melanoma
We initially examined TNFα signaling in response to exogenous TNFα in 40 melanoma cell lines, including 31 cutaneous (11 BRAF-mutant, 10 NRAS-mutant, 10 BRAF/RAS WT)
The loss of pigmentation antigens in response to TNFα is thought to contribute to the resistance to immune checkpoint inhibitors, and it is possible that this diminished antigen repertoire may be compensated for by the induction of antigen presentation molecules
Summary
A total of 40 melanoma cell lines were included in this study, provided by Prof. Peter Parsons at QIMR Berghofer Medical Research Institute (Australia), Prof. Hersey at the Centenary Institute (Australia) and Prof. Park Memorial Institute-1640 media (RPMI) supplemented with 10 or 20% heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), 4 mM glutamine (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 20 mM HEPES (Gibco) and were maintained at 37 ◦ C in 5% CO2. For TNFα treatment, melanoma cells were seeded in. 6-well plates (0.5–1 × cells/well) or in T75 flasks (0.5–1 × cells/flask), and after an overnight incubation, cells were treated for 72 h with 1000 U/mL TNFα (Peprotech, Rocky Hill, NJ, USA) or control (0.1% bovine serum albumin (BSA, Sigma-Aldrich) in phosphate-buffered saline (PBS, Gibco)
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