Modulation of Immune Checkpoints by Tumour Extracellular Vesicles

Shahaj, Eriomina (2020). Modulation of Immune Checkpoints by Tumour Extracellular Vesicles. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.00011212

Abstract

Melanoma extracellular vesicles (EVs) are endowed with pro-tumourigenic features and can condition immunity, favouring immune escape. Immune checkpoints (IC), expressed by immune and cancerous cells, regulate immune responses. BRAF and MEK inhibitors, a standard treatment for BRAF-mutated melanoma patients, can condition tumour cell IC expression. I investigated the IC expression in melanoma specimens, cell lines and EVs, in association with drug resistance, a common event during treatment, to uncover potential targets for immunotherapy. IC were studied in melanoma and immune cells interacting with melanoma EVs from drug sensitive and resistant cell line pairs with CD81GFP-tagged EVs. IC were determined by qRT-PCR, western blot and latex beads flow cytometry on cell line and plasma EVs, isolated by ultracentrifugation and commercial kits. EVs were measured by Nanoparticle Tracking Analysis in whole plasma and after isolation from cell lines. In melanoma lesions CD155, HVEM and GAL9 transcripts displayed a modulation in association with drug resistance. In cell lines, PDL1, PDL2 and CD155 increased, while GAL9 and HVEM decreased with resistance. PDL1, PDL2 and GAL9 modulations were influenced by IFNγ and regulated by CCL2 and specific miRNAs. EVs carried IC and reflected the IC expression pattern of originating melanoma cells. EVs from resistant melanoma cells transferred drug resistance-associated features to sensitive counterparts; while EVs from both, sensitive and resistant melanoma cells induced in monocytes a myeloid-derived suppressor cell phenotype accompanied with the increase of PDL1 and HVEM expression. During therapy patients’ plasma EVs displayed modulations in number and size. Their IC, as protein and transcripts, associated with response to treatment. BRAF and MEK inhibitors modulate melanoma IC expression and cognate EVs reflect originating cells, potentially representing surrogates of melanoma resistance status. The effects of IC-carrying EVs on interacting cells suggest their involvement in tumour immune escape, and their modulations may reflect immune activation during therapy.

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