Separation of plasma-derived exosomes into CD3(+) and CD3(−) fractions allows for association of immune cell and tumour cell markers with disease activity in HNSCC patients

Abstract

Head and neck squamous cell carcinoma (HNSCC) is a highly immunosuppressive malignancy. Exosomes in HNSCC patients' plasma are enriched in inhibitory cargo and mediate immunosuppression. As these exosomes are products of various cells, the cellular origin of immunoregulatory proteins they carry is unknown. To test whether tumour- or T cell-derived exosomes in patients' plasma are immunosuppressive and impact upon disease activity, we separated CD3 from CD3 exosomes by immunocapture using anti-CD3 antibodies. The exosome protein cargo was evaluated for immunoregulatory proteins using on-bead flow cytometry. Tumour protein-enriched CD3 exosomes were CD44v3 . Surprisingly, mean levels of programmed death ligand 1 (PD-L1), cytotoxic T lymphocyte antigen 4 (CTLA-4) and cyclooxygenase-2 (COX-2) were similar in CD3 and CD3 exosomes, although the latter induced higher (P < 0·0025) ex-vivo apoptosis of CD8 T cells and greater (P < 0·005) conversion of CD4 T cells to CD4 CD39 regulatory T cells (T ). CD3 and CD3 exosomes carrying high levels of immunosuppressive proteins were highly effective in mediating these functions. Exosomes of patients with Union for International Cancer Control (UICC) stages III/IV disease had higher levels of PD-L1 and COX-2 than stages I/II patients (P < 0·005). Patients with nodal involvement had exosomes with the higher inhibitory protein content than N0 patients (P < 0·03). CD3 and CD3 exosomes of HNSCC patients had higher PD-L1, COX-2 and CD15s levels than healthy donors' exosomes (P < 0·009), although levels of immunostimulatory OX40 or OX40L were not different. By isolating CD3 /CD44v3-enriched and CD3 exosomes from plasma, the cellular origins of immunoregulatory proteins they carry were identified. Association of exosome molecular profiles with disease progression supports the exosome potential as future cancer biomarkers.

Isolation and Analysis of Tumor‐Derived Exosomes

“…Although exosomes isolated from plasma of cancer patients are enriched in TEX, additional separation and further enrichment in TEX might be obtained by immune capture. T-cell-derived exosomes represent a substantial proportion (up to 50%) of total exosomes isolated from cancer patients' plasma (Theodoraki et al, 2018), and removal of CD3(+) exosomes leaves behind an exosome fraction highly enriched in TEX. The two exosome fractions, one enriched in TEX and the other containing only CD3(+) exosomes, are recovered and characterized.…”

“…Immune capture of CD3(+) exosomes is readily accomplished by using commercially available anti-CD3 monoclonal antibodies (mAbs; Theodoraki et al, 2018). The procedure involves streptavidin-labeled beads coated with biotinylated anti-CD3 mAbs for capture of CD3(+) exosomes, as illustrated in Figure 4.…”

“…They carry a rich cargo of proteins, lipids, glycans, and This protocol describes a series of procedures necessary for isolation of exosomes from supernatants of tumor cell lines or from plasma of cancer patients. Exosomes isolated from supernatants are TEX, while those isolated from plasma or other body fluids are mixtures of exosomes derived from a variety of normal cells but enriched in TEX (Ludwig et al, 2019;Theodoraki, Hoffmann, & Whiteside, 2018). The protocol starts with methods describing the harvesting of tumor cell supernatants, followed by the preclearing of supernatants aimed at the removal of cell debris, apoptotic bodies, and large microvesicles.…”

Exosomal miR‐103a‐3p ameliorates lipopolysaccharide‐induced immune response in BEAS‐2B cells via NF‐κB pathway by targeting transducin β‐like 1X related protein 1

“…23,24 Plasma-derived exosomes have been reported to participate in cell proliferation, apoptosis and immune response. [25][26][27] High expression of exosomal miR-21 was found in serum samples of ventilator-associated pneumonia patients. 28 Overexpression of miR-371b-5p promotes alveolar progenitor type II cell proliferation.…”

“…MiRNAs, as important signalling molecules, abundantly exist in plasma exosomes . Plasma‐derived exosomes have been reported to participate in cell proliferation, apoptosis and immune response . High expression of exosomal miR‐21 was found in serum samples of ventilator‐associated pneumonia patients .…”