Circulating Exosomes Inhibit B Cell Proliferation and Activity.

(1) Background: Head and neck squamous cell carcinoma (HNSCC) is characterized by a distinctive suppression of the anti-tumor immunity, both locally in the tumor microenvironment (TME) and the periphery. Tumor-derived exosomes mediate this immune suppression by directly suppressing T effector function and by inducing differentiation of regulatory T cells. However, little is known about the effects of exosomes on B cells. (2) Methods: Peripheral B cells from healthy donors and HNSCC patients were isolated and checkpoint receptor expression was analyzed by flow cytometry. Circulating exosomes were isolated from the plasma of HNSCC patients (n = 21) and healthy individuals (n = 10) by mini size-exclusion chromatography. B cells from healthy individuals were co-cultured with isolated exosomes for up to 4 days. Proliferation, viability, surface expression of checkpoint receptors, and intracellular signaling were analyzed in B cells by flow cytometry. (3) Results: Expression of the checkpoint receptors PD-1 and LAG3 was increased on B cells from HNSCC patients. The protein concentration of circulating exosomes was increased in HNSCC patients as compared to healthy donors. Both exosomes from healthy individuals and HNSCC patients inhibited B cell proliferation and survival, in vitro. Surface expression of inhibitory and stimulatory checkpoint receptors on B cells was modulated in co-culture with exosomes. In addition, an inhibitory effect of exosomes on B cell receptor (BCR) signaling was demonstrated in B cells. (4) Conclusions: Plasma-derived exosomes show inhibitory effects on the function of healthy B cells. Interestingly, these inhibitory effects are similar between exosomes from healthy individuals and HNSCC patients, suggesting a physiological B cell inhibitory role of circulating exosomes.

Immune Suppressive Effects of Plasma-Derived Exosome Populations in Head and Neck Cancer.

Plasma-derived exosomes of head and neck squamous cell carcinoma (HNSCC) patients carry inhibitory factors mediating immune suppression. Separation of tumor-derived exosomes (TEX) and non-TEX may assist in a better understanding of their respective parental cells. Here, we evaluate the impact of TEX or hematopoietic-derived exosomes on immune suppression. We evaluated apoptosis in CD8+ T cells, conversion of CD4+ T cells to regulatory T cells (Treg), and adenosine production by TEX, non-TEX, or total exosomes. Exosome protein cargo was significantly higher in total and CD45(-) exosomes from high stage compared to low stage patients. Furthermore, total and CD45(-) exosomes of high stage patients induced more apoptosis in CD8+ T cells than their low stage counterparts. CD69 suppression, a marker of reduced CD8+ T cell activation, was only mediated by CD45(-) exosomes. All fractions induced Treg differentiation, defined by CD39 expression, but only CD45(-) exosomes showed a stage-dependent conversion. CD45(-) exosomes produced higher adenosine concentrations than CD45(+) exosomes, concluding that adenosine production measured in total exosomes mainly derives from TEX. The presented results show significant induction of immune suppression by TEX in HNSCC. This immunosuppressive effect supports the potential role of exosomes as liquid biomarkers for disease stage and level of immune suppression.

CD44v3 protein-carrying tumor-derived exosomes in HNSCC patients' plasma as potential noninvasive biomarkers of disease activity.

The molecular cargo of tumor-cell-derived exosomes (TEX) mimics that of parental tumor cells. Thus, TEX could potentially serve as noninvasive biomarkers of cancer progression. However, separation of TEX from non-TEX in patients' plasma requires tumor antigen-specific detection reagents. CD44v3 has been of interest as a potential biomarker of disease progression in HNSCC, because its overexpression in tumor cells associates with poor outcome. Here, CD44v3+ TEX immunocaptured from plasma of 44 HNSCC patients and 7 healthy donors (HDs) were evaluated as potential biomarkers of disease activity and stage. Exosomes were isolated from plasma of by size exclusion chromatography. Using anti-CD44v3 or anti-CD3 mAbs on beads, CD44v3+ TEX CD3(-)TEX-enriched exosomes were immunocaptured from supernatants of nonmalignant or HNSCC cell lines and from patients' plasma. On-bead flow cytometry was used for the detection of FAS-L, PD-L1, TGFF-ß. CSPG4 or EGFR on exosomes. The TEX expression profiles were correlated to clinicopathological parameters. Relative florescence intensity (RFI) values for CD44v3 were higher (p < .01) on TEX from HNSCC cell lines or on CD44v3+ CD3(-) plasma-derived exosomes. RFI values of CD44v3 on CD3(-) exosomes were higher (p < .005) in patients than in HDs and correlated (p < .05) with the UICC stage and lymph node metastasis. In HNSCC patients, CD44v3+ exosomes higher levels of immunosuppressive proteins compared to CD44v3(-) exosomes (p < .05-p < .005), and RFI values for these markers correlated with higher disease stages and lymph node metastasis. Isolation of CD44v3+ exosomes by immunocapture allowed for enrichment of TEX which are potentially promising liquid biomarkers of the tumor burden and disease stage in HNSCC.