Human tumor-derived exosomes (TEX) regulate Treg functions via cell surface signaling rather than uptake mechanisms

Abstract

Tumor-derived exosomes (TEX) are ubiquitously present in the tumor microenvironment and plasma of cancer patients. TEX carry a cargo of multiple stimulatory and inhibitory molecules and deliver them to recipient cells, serving as a communication network for the tumor. The mechanisms TEX use for delivering messages to recipient cells were evaluated using PKH26-labeled TEX produced by cultured human tumor cells, exosomes produced by dendritic cells-derived exosomes (DEX), or exosomes isolated from plasma of cancer patients (EXO). Human T-cell subsets, B cells, NK cells, and monocytes were co-incubated with TEX, DEX, or EXO and binding or internalization of labeled vesicles was evaluated by confocal microscopy and/or Amnis-based flow cytometry. Vesicle-induced Ca²⁺ influx in recipient T cells was monitored, and TEX-induced inosine production in Treg was determined by mass spectrometry. In contrast to B cells, NK cells or monocytes, conventional T cells did not internalize labeled vesicles. Minimal exosome uptake was only evident in Treg following prolonged co-incubation with TEX. All exosomes induced Ca²⁺ influx in T cells, with TEX and EXO isolated from cancer patients' plasma delivering the strongest, sustained signaling to Treg. Such sustained signaling resulted in the significant upregulation of the conversion of extracellular ATP to inosine (adenosine metabolite) by Treg, suggesting that TEX signaling could have functional consequences in these recipient cells. Thus, modulation of Treg suppressor functions by TEX is mediated by mechanisms dependent on cell surface signaling and does not require TEX internalization by recipient cells.

Keywords: Ca2+ flux; T cell subsets; exosome uptake; regulatory T cells; tumor-derived exosomes.

Figure 1.

Differential uptake of PKH26-labeled TEX by U937tumor cells and by Jurkat T cells. Representative images showing strong internalization of PKH26-labeled TEX by U937 cells (upper row) but no TEX uptake in CD8⁺ Jurkat cells (lower row). TEX were isolated from supernatants of PCI-13 tumor cells and labeled with the PKH26 dye as described in Methods. Cells co-incubated with 10 µg of TEX for 24 h in a 35 mm MatTek plate were examined in a confocal microscope. The bar = 5 μm.

Figure 2.

Differences in uptake of PKH26-labeled TEX cells and monocytes. (A) Time-dependent uptake of TEX by monocytes contrasts with the slower and lower TEX uptake by T cells. Amnis measurements of PKH26 intensity in cells were acquired after increasing co-incubation times. (B) Representative Amnis images show TEX uptake by monocytes vs. no uptake by T cells after 48 h of co-incubation.

Figure 3.

Amnis-generated data showing differences in TEX uptake by various human MNC subsets. (A) Various MNC subsets isolated from different ND were co-incubated with TEX for 24–48 h. T cells T show significantly lower TEX uptake compared with other MNC subsets. (B) Comparisons of TEX uptake by resting or activated CD8⁺ T cells, CD4⁺ T cells, or CD4⁺CD39⁺ Treg show a lack of TEX internalization by CD8⁺ T cells relative to low but significantly increased TEX uptake by Treg. T cell subsets were isolated from the peripheral blood of four different ND. In (A) and (B), the data are presented as mean levels of PKH26 intensity in recipient cells ± SD. The p values denote significant differences.

Figure 4.

Amnis-generated representative images of recipient MNC co-incubated with PKH26-labeled TEX for 48 or 72 h. Immune cell subsets were isolated from healthy donors' plasma and analyzed by Amnis Image Stream as described in Methods. The presented images are representative results of four experiments performed with MNC of different donors and show results obtained by a triple overlay (PKH26-stain in yellow, surface stain in red, and a brightfield image) as described in Methods.

Figure 5.

Ca²⁺ flux in human T cells co-incubated with TEX, DEX, or EXO. In (A), T cells co-incubated with exosomes were labeled with Fluo-3 and examined in a Nikon A1 microscope as indicated in Methods. Ionomycin, which induces strong Ca²⁺ flux, was used as a positive control. T cells were imaged at different times after Fura-2 addition. The upper panel compares effects of TEX and DEX. The lower panel compares effects of EXO isolated from plasma of a representative HNSCC patient vs. EXO isolated from ND's plasma. In (B), Fluo-3 intensity levels in T cells co-incubated with TEX or DEX. The data are from a representative experiment of 3 performed with T cells of different donors. Note the strong and prolonged signal in T cells co-incubated with TEX and the absence of signaling in T cells co-incubated with DEX. In C, Fluo-3 intensity levels in CD4⁺T conv cells co-incubated with EXO from the plasma of HNSCC patient with active disease (AD) or with EXO from ND's plasma. Note the strong, prolonged signal induced by patients' EXO vs. an absence of signal with ND's EXO. Shown are representative Ca²⁺ flux data obtained from four to six experiments performed with T cells of different subjects.

Figure 6.

Mass spectrometry for purine derivatives of adenosine in co-cultures of TEX with Treg or CD4⁺ T cells. TEX were co-incubated with T cells or PCI-13 tumor cells in the presence of exogenous ATP for 16 h. T cells were isolated from three different donors. Cells and supernatants were collected and prepared for measurements of purines by mass spectrometry as described in Methods. In (A), TEX but not the parent PCI-13 tumor cells induced a large increase in inosine production by CD4⁺CD39⁺ Treg. In (B), TEX co-incubated with CD4⁺T cells also induced the upregulation of inosine production, although there were large differences among cells of different donors. The data are means ± SEM from experiments performed with T cells of three different donors.