Effects of the interaction of EVs with immune cells

In the experimental part of the project that investigated the effects of melanoma EV on immune blood cells, I showed the EV uptake by monocytes beside the modulation of PDL1, CD155, HVEM and GAL9 expression. Indeed, EV-interacting monocytes acquire phenotypic and functional properties of MDSC, as already demonstrated from the hosting lab.

Experiments showed that melanoma EVs interacted almost exclusively with the CD14+

monocyte fraction in PBMCs of healthy donors, whether obtained from both BRAF/MEKi

sensitive or resistant cells lines. In fact, I could observe no interaction with CD3 T cells of FITC positive EVs. The preferential targeting of monocytes has been demonstrated by many studies, thus it represented an expected result [270]. In contrast to recent studies reporting EV interaction with T cells majorly by surface-surface receptor-ligand interaction [115, 271], my data showed no EV internalisation or interaction with T cells in the co-culture experiments. However, an EV-mediated triggering of surface molecules expressed by T cells is very likely to occur. EVs that bound to T cells potentially detached when I harvested the cell samples for flow cytometry labelling and acquisition.

Monocyte-EV interaction detected after 18h co-culture was accompanied by an upregulation of PDL1 that was particularly evident in those monocytes which internalized

EVs and were detected either as GFP positive or as DIOC-FITC positive monocytes (Figure 4.1.1). Similarly, purified CD14+ _{cells cultured in the presence of GFP-tagged EVs displayed}

an acquisition of green fluorescence and an increase of the percentage of CD14+_PDL1+

cells. The induction of PDL1 expression in monocytes was previously described in CLL patients as mediated by tumour exosome-carried non coding RNA, which targeted TLR7 in monocytes [272]. In their work, Haderk et al. also observed a production of proinflammatory cytokines CCL2, CCL4 and IL-6 similarly to what we detected upon co- culture of melanoma EVs in autologous and healthy donors’ monocytes [62]. A recent study showed that PDL1 is upregulated by monocytes upon interaction with glioblastoma stem cell-derived exosomes. Interestingly, despite these authors observed a preferential

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interaction with monocytes, they also detected an uptake of these EVs by activated but not resting T cells at 6 h of incubation [271]. Additionally, Gabrusiewicz and co-workers observed that PDL1 upregulation correlated with STAT3 phosphorylation, a hallmark of MDSC [273], occurring by melanoma EVs expressed HSP86 and TLR4 expressed MDSC interaction [63].

In my studies EVs obtained from both BRAF/MEKi sensitive and resistant cell lines induced a downregulation of HLA-DR expression by monocytes that was evident in gated CD14+

cells in PBMCs as well as after purification. Of note, in the monocyte gate of PBMCs the

decrease of HLA-DR coincided with an upregulation of HVEM on the HLA-DRneg

subpopulation. HVEM expression by MDSC may depend on an endogenous regulation by

the monocytes in response to EV internalisation. In fact HVEM was described as a protein that can be expressed by monocytic MDSC [274]. In my experimental setting, the increased HVEM expression could also depend on the transfer of HVEM transcripts, which I could detect in EVs of the cell line pairs selected for these studies.

In contrast with data in the literature depicting that patients’ MDSC express increased levels of GAL9, my results showed a downregulation of this IC on the HLA-DRneg_HVEMhigh

as compared to the HLA-DRhigh_HVEMlow _{monocytes [59]. This discrepancy could rely on} the different settings, e.g. ex vivo patients’ monocytes vs in vitro experiments. In fact, another study investigating the generation of MDSC in an in vitro setting described a reduced expression of GAL9 by M-MDSC [274]. Regarding PDL1 and PDL2, my experiments showed the induction of membrane expression of these IC on monocytes after the short-term incubation of 18 and 24 h, while at 36 h a minor but statistically significant reduction was detected. At opposite, PDL1 and PDL2 transcript levels always displayed an increase, in healthy donors’ monocytes by both EVs from resistant and sensitive melanoma cell lines and in the autologous setting with either melanoma and plasma-derived EVs. This coincided with an increase of CD163, a M2 marker of macrophages associated with cancer progression through induction of IL-6 expression [274]. Interestingly, IL-6 and CCL2 were the main cytokines I found upregulated in monocytes cultured with melanoma EVs, as well as when EV-MDSC were induced by MDSC-microRNAs carried by EVs [62].

To test the suppressive ability of MDSC induced by melanoma EVs in monocytes from healthy donors’ and to assess potential differences between EVs deriving from sensitive vs drug resistant melanoma cell lines, I performed proliferation experiments at different

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monocytes ratios. In the absence of EVs conditioning, monocytes inhibited T cell proliferation if present at high ratios in co-cultures (100% and 50%), while at 25% and at 12.5% they stimulated proliferation of both CD4 and CD8 T cells. This is in line with previous findings showing that in the absence of disease high ratios of monocytes can influence T cell activity [275]. The suppressive activity of EVs-conditioned monocytes was evident already at 25% monocytes and affected both CD4 and CD8 T cells. At the higher ratios of 50% and 100%, the proliferation decreased progressively and at 100% CD8 T cells showed no proliferation. These results showed that melanoma EVs induced a

suppressive phenotype in monocytes, and that no differences could be observed between the origin of EVs, from sensitive melanoma cell line LM16S or fromLM16R cell

line. Additionally, the suppressive activity of monocytes could be also induced by CD81- and PDL1-GFP-tagged LM16S EVs, indicating this setting as an in vitro model lacking labelling biases suitable for studies investigating EV-mediated effects.