Melanoma-associated fibroblasts impair CD8+\u2009T cell function and modify expression of immune checkpoint regulators via increased arginase activity

Barbara Érsek,Eva Mezey,Ugur Cakir,Krisztián Németh,Viktor Molnár,Zoltán Pós,András Bencsik,Balázs Mayer,Pálma Silló,Sarolta Kárpáti

doi:10.1007/s00018-020-03517-8

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.Graphic abstract

Highlights

Cancer-associated fibroblasts (CAF) represent a heterogeneous cell population, considered to originate from various possible precursors, such as resting resident fibroblasts, bone marrow-derived mesenchymal stem cells, hematopoietic stem cells, endothelial cells, local epithelial cells, or adipocytes [1]
We examined whether melanoma-associated fibroblasts (MAFs)-conditioned media (CM) would affect expression of either CD69, an early T cell activation marker, or that of CD107a (LAMP-1), a marker of cytotoxic degranulation
We found that CD8+ T cells activated by anti-CD3/28 in the presence of MAF-CM displayed less CD69 compared to CD8+ T cells activated in the presence of dermal fibroblasts (DFs)-CM, as the frequency of CD69+ cells was decreased

Summary

Introduction

Cancer-associated fibroblasts (CAF) represent a heterogeneous cell population, considered to originate from various possible precursors, such as resting resident fibroblasts, bone marrow-derived mesenchymal stem cells, hematopoietic stem cells, endothelial cells, local epithelial cells, or adipocytes [1]. CAFs are known to suppress local immune responses via a wide array of cytokines, small molecular mediators and metabolic enzymes, such as transforming growth factor beta (TGFβ), IL-6, prostaglandin E2 (PGE2), VEGF and indoleamine-2,3-dioxygenase (IDO)/kynurenine [2, 3]. The relevance of CAFs in local immunosuppression is underlined by multiple observations showing that their removal may prolong survival or achieve immune-mediated rejection of established tumors [4,5,6,7]. It is well documented that CAF (melanoma-associated fibroblasts, MAF) are rather capable suppressors of NKcell activity via multiple ways, including PGE2-dependent suppression [8,9,10], and this phenomenon is probably not restricted to melanoma [11, 12]

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