Critical Role of Hodgkin Lymphoma Cells-Derived Large and Small Extracellular Vesicles in the Regulation of the Immune-Inflammatory Microenvironment

Introduction: Classical Hodgkin Lymphoma (cHL) is characterized by a low number of CD30+ malignant cells and an extensive immune-inflammatory microenvironment. However, this unique histopathological picture and its pathogenesis are still partially understood. Extracellular vesicles (EVs) are submicron particles delimited by a lipid bilayer that promote (tumor) intercellular communication and carry bioactive molecules that can influence the immune system. Based on size and biogenesis mechanism, Small (S-) and Large (L-) EVs (30-200 nm and 0.2-10 µm, respectively) have been identified. Extracellular Adenosine Triphosphate (eATP), a key component of the tumor microenvironment, plays a role in biogenesis and dynamics of EVs. Here we aim to elucidate the role of cHL cell-derived EVs in the education of the immune microenvironment.

Methods: cHL L-428 cell line was cultured in exosome-free medium for 48 hours at 37°C in the presence or absence of eATP (1 mM). L-428 cell blebbing has been detected by fluorescence microscopy (DAPI and FITC-Cholera Toxin B subunit (CTxB) labeling). L-EVs and S-EVs were isolated from the cell culture supernatant by differential ultracentrifugation (2.800g for 2.8K and 20.000g for 20K L-EVs, and 100.000g for 100K S-EVs) and purified by iodixanol density gradient. L- and S-EVs were then characterized by Tunable Resistive Pulse Sensing (TRPS), single EV-flow cytometry, and MACSPlex (bead-based antigen binding array). Functionally, normal peripheral blood mononuclear cells were in vitro cultured with titrated amount of isolated L- and S-EVs (from 1:5 to 1:20) for 24 hours. The intracytoplasmic interleukin-1β and interferon-γ production by CD14+ monocytes and CD3+/CD4+/CD8+ T cells was assessed by flow cytometry.

Results: Our results showed that human cell line L-428 is able to produce L-EVs through membrane blebbing (Figure 1A). According to EV size distributions, both 2.8K and 20K L-EVs displayed a size range from 1 to 4 µm whereas S-EVs were enriched in particles of 150-200 nm diameter. Interestingly, the amounts of S-EVs shed by L-428 cells were significantly higher compared to the number of L-EVs. Considering the EV surface protein phenotype, S-EVs express all the EV-related tetraspanin proteins (CD9, CD63, and CD81). By contrast, L-EV express CD63 and CD81 only. Additionally, in L-EVs we observed an enrichment of cancer-related markers including SSEA-4, CD44, CD24, CD29 and CD133-1 in comparison with the S-EV counterpart. Of note, the expression of selected immune-related molecules such as CD86 and CD40 was higher in the 2.8K and 20K L-EVs as compared with that of the 100K S-EVs. The HLA-II molecules were expressed by all fractions, with the highest expression in 2.8K L-EVs. Along with PD-L1, the CD30 HL-associated marker was expressed by all EV fractions at the same extent. When we tested the effects of eATP on EVs produced by L-428 cells, we found a highly decline in the expression of EV-surface proteins from all EV sub-fractions, being most noticeable in L-EVs. Importantly, only PD-L1 expression was increased in the 20K L-EVs after eATP treatment. Functionally, at variance with S-EVs (Figure 1B), L-EVs highly stimulate the intracytoplasmic production of interleukin-1β by monocytes. Conversely, L-EVs derived from e-ATP treated L-428 cells fail to stimulate the intracytoplasmic production of interleukin-1β by CD14+ monocytes, which instead was promoted by the e-ATP treated S-EV counterparts. Of note, neither L-EVs nor S-EVs stimulated interferon-γ production by CD14+ monocytes. No intracytoplasmic production of the selected inflammatory cytokines has been observed when T-cells treated with EVs were analyzed.

Conclusions: These results demonstrate that L-EVs from HL cells display a cancer- and immune-related phenotype and play a role in the modulation of the immune microenvironment by promoting the production of inflammatory cytokines by monocytes. This might be partially due to the high expression of CD44 on the surface of L-EVs, since it has been previously demonstrated that cancer-derived soluble CD44 may trigger monocyte/macrophage interleukin-1β production. Interestingly, eATP plays a role in shaping the immune modulatory ability of L- and S-EVs. These findings shed light on the role of HL-derived EVs in the establishment of the immune-inflammatory microenvironment and provide novel insight into clinically relevant EV driven mechanisms in HL.

Casadei:Gilead-Science: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Bristol Mayers Squibb: Consultancy; BeiGene: Consultancy; Incyte: Consultancy; Abbvie: Consultancy. Zinzani:Beigene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSA Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sandoz: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Secura Bio: Membership on an entity's Board of Directors or advisory committees.