Suppression of Lymphocyte Functions by Plasma Exosomes Correlates with Disease Activity in Patients with Head and Neck Cancer

Abstract

Purpose: Head and neck cancers (HNCs) often induce profound immunosuppression, which contributes to disease progression and interferes with immune-based therapies. Body fluids of patients with HNC are enriched in exosomes potentially engaged in negative regulation of antitumor immune responses. The presence and content of exosomes derived from plasma of patients with HNC are evaluated for the ability to induce immune dysfunction and influence disease activity.Experimental Design: Exosomes were isolated by size-exclusion chromatography from plasma of 38 patients with HNC and 14 healthy donors. Morphology, size, numbers, and protein and molecular contents of the recovered exosomes were determined. Coculture assays were performed to measure exosome-mediated effects on functions of normal human lymphocyte subsets and natural killer (NK) cells. The results were correlated with disease stage and activity.Results: The presence, quantity, and molecular content of isolated, plasma-derived exosomes discriminated patients with HNC with active disease (AD) from those with no evident disease (NED) after oncologic therapies. Exosomes of patients with AD were significantly more effective than exosomes of patients with NED in inducing apoptosis of CD8⁺ T cells, suppression of CD4⁺ T-cell proliferation, and upregulation of regulatory T-cell (Treg) suppressor functions (all at P < 0.05). Exosomes of patients with AD also downregulated NKG2D expression levels in NK cells.Conclusions: Exosomes in plasma of patients with HNC carry immunosuppressive molecules and interfere with functions of immune cells. Exosome-induced immune suppression correlates with disease activity in HNC, suggesting that plasma exosomes could be useful as biomarkers of HNC progression. Clin Cancer Res; 23(16); 4843-54. ©2017 AACR.

Figure 1. Protein levels in exosomes isolated from plasma of HNC patients or NDs

Following mini-SEC isolation of exosomes in fraction #4, total exosomal protein was measured in BCA assays. (A) Exosome protein levels are significantly higher in AD compared to NED and ND (p<0.01), whereas exosome protein levels in NED and ND are similar. (B) In AD patients, exosomes from plasma of advanced HNCs (UICC stage III/IV) have significantly higher protein levels than those from early stage diseases (UICC stage I/II) (p<0.001). (C) Following surgery alone, NED patients’ exosomes tend to have lower protein levels than exosomes from plasma of patients treated with surgery plus adjuvant oncotherapy. The data in A-C are mean values ±SEM.

Figure 2. Size distributions and particle concentrations in exosome #4 fractions isolated from plasma of HNC patients and NDs

In (A), representative data obtained with fraction #4 exosomes from AD, NED or ND plasma show the same size distribution, while numbers of the recovered particles are highest in the AD #4 fraction. In (B), the combined data for 3 different exosome donors in each cohort are shown. The mean particle concentration is highest in AD patients, intermediate in NED and lowest in NDs (**AD vs. ND p<0.01; * NED vs. ND p<0.05). The data are mean values ± SEM.

Figure 3. Transmission electron microscopy (TEM) images of exosomes isolated from plasma of HNC patients and a ND

Exosomes appear as vesicles ranging in size from 30 to 100nm. Exosome morphology is the same for AD and NED patients and the ND. The shown images are representative for 1/5 exosome fractions examined for each cohort.

Figure 4. Molecular protein profiles of plasma exosomes from AD and NED patients and a ND

Western blots were performed after loading 10μg of exosomal protein/lane. Immunosuppressive in (A) and immunostimulatory in (B) protein profiles are shown for exosomes of 4 patients with AD, 4 patients with NED and of one ND. In (C) PRAME is used as a marker for HNC, and TSG101 serves as an exosome marker.

Figure 5. Exosomes-mediate apoptosis in CD8⁺ Jurkat cells or primary activated CD8+ T cells

In (A), representative flow cytometry data for Annexin V binding in CD8⁺ Jurkat cells co-incubated with exosomes isolated from HNC patients or a ND. In (B), exosomes of AD patients carry higher FasL than exosomes of NED (NSD) and AD exosomes induce significantly more apoptosis than NED exosomes (p<0.05). In (C), flow cytometry data for apoptosis of primary activated CD8⁺ T cells co-incubated with plasma-derived exosomes for 24h. There was a significant correlation between T-cell apoptosis with the patients’ disease activity (*p<0.05, **p<0.01, ****p<0.0001). In (D), combined data (means ± SEM) for caspase 3/7 activation in primary CD8⁺ T cells co-incubated for 24h with exosomes as described above (*p<0.05).

Figure 6. Exosome-mediated suppression of CD4⁺ T cell proliferation as measured in a CFSE-based assays

Activated CD4⁺ T cells were cultured alone or in the presence of plasma-derived exosomes. In (A), representative data of proliferating CFSE⁺ primary CD4+ T cells co-incubated with exosomes from plasma of HNC patients and a ND. Suppression of proliferation was calculated relative to proliferation without exosomes added. In (B), exosomes from plasma of AD patients suppress proliferation of CD4⁺ T cells significantly better than exosomes of NED patients or exosomes of ND (*p<0.05). Suppression mediated by NED exosomes was comparable to that seen with ND exosomes.

Figure 7. Exosomes from plasma of HNC patients up-regulate CD39 expression levels and adenosine production by Treg

In (A), representative flow cytometry data showing up-regulation of CD39 expression levels in resting CD4⁺ T cells isolated from PBMC of NDs. In (B), data for up-regulation of CD39 by exosomes obtained from plasma of AD, NED or ND after 24h co-incubation. Only exosomes of AD patients significantly augmented CD39 expression levels (*p<0.05). In (C), increased adenosine production by resting CD4⁺CD39⁺ Treg following co-incubation with exosomes from plasma of AD and NED patients or ND in the presence of 20μM exogenous ATP for 1h. Extracellular adenosine levels are the highest in Treg co-incubated with exosomes of AD patients (*p<0.05), intermediate in Treg co-incubated with exosomes of NED patients (*p<0.05) and lowest in Treg co-incubated with exosomes of NDs. The data are from 3 independent experiments of which 2 were performed with duplicate co-cultures.

Figure 8. Exosome-induced down-regulation of NKG2D and suppression of NK cell-mediated cytotoxicity

In (A), representative flow cytometry data for down-regulation of NKG2D expression levels in normal human NK cells by HNC patients’ exosomes. Exosomes were co-incubated with resting PBMCs and gates were set on CD3⁻CD56⁺ NK cells. Exosomes of the AD patient induced the most prominent loss of NKG2D expression levels relative to NED or ND exosomes. In (B) combined MFI data for NKG2D down-regulation by exosomes of HNC patients and NDs are shown. In (C), cytotoxicity of NK cells against K562 targets was measured using CFSE-labeled K562 target cells by flow cytometry. Only exosomes from AD patients suppressed NK cell-mediated cytotoxicity (*p<0.05).