Exosomal CD73 from serum of patients with melanoma suppresses lymphocyte functions and is associated with therapy resistance to anti-PD-1 agents

Abstract

BackgroundCD73 is an ectonucleotidase producing the immunosuppressor mediator adenosine. Elevated levels of circulating CD73 in patients with cancer have been associated with disease progression and poor response to immunotherapy. Immunosuppressive pathways associated with exosomes can affect T-cell function and the therapeutic efficacy of anti-programmed cell-death protein 1 (anti-PD-1) therapy. Here, we conducted a retrospective pilot study to evaluate levels of exosomal CD73 before and early during treatment with anti-PD-1 agents in patients with melanoma and its potential contribution to affect T-cell functions and to influence the clinical outcomes of anti-PD-1 monotherapy.MethodsExosomes were isolated by mini size exclusion chromatography from serum of patients with melanoma (n=41) receiving nivolumab or pembrolizumab monotherapy. Expression of CD73 and programmed death-ligand 1 (PD-L1) were evaluated on exosomes enriched for CD63 by on-bead flow cytometry. The CD73 AMPase activity was evaluated by mass spectrometry, also in the presence of selective inhibitors of CD73. Interferon (IFN)-γ production and granzyme B expression were measured in CD3/28 activated T cells incubated with exosomes in presence of the CD73 substrate AMP. Levels of CD73 and PD-L1 on exosomes were correlated with therapy response. Exosomes isolated from healthy subjects were used as control.ResultsIsolated exosomes carried CD73 on their surface, which is enzymatically active in producing adenosine. Incubation of exosomes with CD3/28 activated T cells in the presence of AMP resulted in a significant reduction of IFN-γ release, which was reversed by the CD73 inhibitor APCP or by the selective A2A adenosine receptor antagonist ZM241385. Expression levels of exosomal CD73 from serum of patients with melanoma were not significantly different from those in healthy subjects. Early on-treatment, expression levels of both CD73 and PD-L1 on exosomes isolated from patients receiving pembrolizumab or nivolumab monotherapy were significantly increased compared with baseline. Early during therapy exosomal PD-L1 increased in responders, while exosomal CD73 resulted significantly increased in non-responders.ConclusionsCD73 expressed on exosomes from serum of patients with melanoma produces adenosine and contributes to suppress T-cell functions. Early on-treatment, elevated expression levels of exosomal CD73 might affect the response to anti-PD-1 agents in patients with melanoma who failed to respond to therapy.

Emerging roles of purinergic signaling in anti-cancer therapy resistance

“…Frontiers in Cell and Developmental Biology frontiersin.org melanoma, CD73 is expressed also on exosomes from serum of patients and contributes to lymphocyte T functions suppression and influences the response to anti-PD1 therapy (Turiello et al, 2022). Blocking of both CD73 and A2AR adenosine signaling at the same time, reduces tumor growth and metastasis and improves antitumor immune responses (Young et al, 2016).…”

“…The deficiency of CD73 in CD8 + T cells leads to an increase of IFN-γ, TNFα, granzime B production and mitochondrial respiration, meaning that this ectonucleotidase restraints CD8 + T cells metabolic fitness ( Briceño et al, 2021 ) ( Figure 2B ). In melanoma, CD73 is expressed also on exosomes from serum of patients and contributes to lymphocyte T functions suppression and influences the response to anti-PD1 therapy ( Turiello et al, 2022 ). Blocking of both CD73 and A2AR adenosine signaling at the same time, reduces tumor growth and metastasis and improves antitumor immune responses ( Young et al, 2016 ).…”

Molecular Docking and Intracellular Translocation of Extracellular Vesicles for Efficient Drug Delivery

“…[ 164 , 165 , 166 , 167 , 168 , 169 , 170 , 171 , 172 , 173 , 174 , 175 , 176 , 177 , 178 ]. PD-L1 is present in exosomes released from melanoma cells [ 178 , 179 , 180 , 181 , 182 , 183 , 184 , 185 , 186 ]; however, exosomes derived from metastasised melanoma cells have a higher PD-L1 content than those derived from primary focal melanoma cells. Moreover, an electron microscope analysis revealed the PD-L1 protein was carried on the surface of the protein [ 187 ].…”

Proteomics, Phosphoproteomics and Mirna Analysis of Circulating Extracellular Vesicles through Automated and High-Throughput Isolation

“…By transporting nucleic acids, proteins, lipids, amino acids, and metabolites, EVs play an important role in facilitating remote signal transmission in an organism, which is vital to the multi-organ collaboration and maintenance of normal physiological functions [4,5]. Many physiological processes have been discovered to be related to the EVs [6][7][8][9]. For example, Grizzle et al found that tumor-derived vesicles could bear immunosuppressive molecules, inactivating T lymphocytes or natural killer cells or promoting the differentiation of regulatory T lymphocytes or myeloid cells to suppress immune responses [10].…”