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  • Melanoma-associated fibroblasts impair CD8+ T cell function and modify expression of immune checkpoint regulators via increased arginase activity.

Melanoma-associated fibroblasts impair CD8+ T cell function and modify expression of immune checkpoint regulators via increased arginase activity.

Cellular and molecular life sciences : CMLS (2020-04-25)
Barbara Érsek, Pálma Silló, Ugur Cakir, Viktor Molnár, András Bencsik, Balázs Mayer, Eva Mezey, Sarolta Kárpáti, Zoltán Pós, Krisztián Németh
ABSTRACT

This study shows that melanoma-associated fibroblasts (MAFs) suppress cytotoxic T lymphocyte (CTL) activity and reveals a pivotal role played by arginase in this phenomenon. MAFs and normal dermal fibroblasts (DFs) were isolated from surgically resected melanomas and identified as Melan-A-/gp100-/FAP+ cells. CTLs of healthy blood donors were activated in the presence of MAF- and DF-conditioned media (CM). Markers of successful CTL activation, cytotoxic degranulation, killing activity and immune checkpoint regulation were evaluated by flow cytometry, ELISPOT, and redirected killing assays. Soluble mediators responsible for MAF-mediated effects were identified by ELISA, flow cytometry, inhibitor assays, and knock-in experiments. In the presence of MAF-CM, activated/non-naïve CTLs displayed dysregulated ERK1/2 and NF-κB signaling, impeded CD69 and granzyme B production, impaired killing activity, and upregulated expression of the negative immune checkpoint receptors TIGIT and BTLA. Compared to DFs, MAFs displayed increased amounts of VISTA and HVEM, a known ligand of BTLA on T cells, increased L-arginase activity and CXCL12 release. Transgenic arginase over-expression further increased, while selective arginase inhibition neutralized MAF-induced TIGIT and BTLA expression on CTLs. Our data indicate that MAF interfere with intracellular CTL signaling via soluble mediators leading to CTL anergy and modify immune checkpoint receptor availability via L-arginine depletion.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Human Serum, from human male AB plasma, USA origin, sterile-filtered
Sigma-Aldrich
Arginase Activity Assay Kit, sufficient for 200 colorimetric tests
Sigma-Aldrich
BEC hydrochloride, ≥98% (HPLC)
Sigma-Aldrich
Penicillin-Streptomycin, Solution stabilized, with 10,000 units penicillin and 10 mg streptomycin/mL, 0.1 μm filtered, BioReagent, suitable for cell culture
SAFC
L-Glutamine Solution 200 mM, 29.23 mg/mL in saline, solution, suitable for cell culture
Sigma-Aldrich
Histopaque®-1077, sterile-filtered, density: 1.077 g/mL