To determine the expression of CD73 by flow cytometry, purified exosomes were captured for CD63 and bound to magnetic beads. Expression of CD73 on isolated exosomes was evaluated using a specific antibody anti CD73 in comparison with the isotype control . CD73 was detected on exosomes isolated from healthy subjects and patients with melanoma, with no significant difference between the two groups . In this study, we used serum from patients with metastatic melanoma receiving anti-PD-1 agents, nivolumab or pembrolizumab, umarkets forex broker review to isolate exosomes and evaluate the functional relevance of exosomal CD73 in affecting T-cell functions ex vivo and as a potential biomarker of response to immunotherapy. In line with published data,10 the expression levels of exosomal PD-L1 resulted significantly increased at week 4 in the responders group but not in non-responders . Altogether, these results suggest that changes in CD73 levels in circulating exosomes early during therapy would reflect a failure to respond to anti-PD-1 agents.
CD73 expressed on exosomes from serum of patients with melanoma produces adenosine and contributes to suppress T-cell functions. Early on-treatment, elevated expression levels of exosomal CD73 might affect the response to anti-PD-1 agents in patients with melanoma who failed to respond to therapy. Previous studies have demonstrated that cancer exosomes express CD73.23–25 Analysis by western blotting indicated the presence of CD73 on isolated exosomes .
The mean fluorescence intensity of positive beads, gated on forward and sideward light scatter, was measured. Results are expressed as relative fluorescence intensity, calculated as ratio between the MFI of the target of interest and the MFI of the control isotype. Vesicle size distribution was measured by dynamic light scattering using a Zetasizer V.7.01, Malvern Instrument . Each sample was dispersed in deionized water and the intensity of the scattered light was measured with a detector at 90° angle at room temperature.
In addition, this latter effect resulted abrogated in presence of the CD73 inhibitor APCP . Notably, treatment of cells with exosomes alone did not influence the production of IFN-γ in CD3/28 activated cells in comparison with activated control cells , ruling out any possible direct effects of exosomes on IFN-γ production in our experimental conditions. Furthermore, we also verified that, in our experimental conditions, treatment with melanoma exosomes, in presence or not of AMP, did not affect the cells viability . pf derivatives: broker review On the other hand, considering the crucial role of CD73 in generating the potent immunosuppressor adenosine, our results reinforce the therapeutic potential of targeting CD73. We investigated the functional relevance of CD73 expressed on exosomes isolated from serum of patients with melanoma receiving nivolumab or pembrolizumab monotherapy. This study is the first to report an association between exosomal CD73 expression levels and patient responses to anti-PD-1 agents early during treatment of metastatic melanoma.
Producing adenosine, CD73 potently suppresses the function of T cells.36 To test the effect of exosomal CD73 on T-cell functions, we used human peripheral blood cells from healthy donors. T cells were activated with an anti CD3/CD28 T cell activator, in presence or not of exosomes derived from patients with melanoma and the CD73 substrate AMP. We measured the IFN-γ levels in the supernatants of cells 72 hours later treatment by ELISA. The results show that the levels of IFN-γ in cells pre-treated with exosomes were significantly reduced when adding the substrate AMP compared with control or activated cells treated with exosomes alone .
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Beads-assisted flow cytometry
Please declare your traffic by updating your user agent to include company specific information. This study was approved by the Ethics Committee of Istituto Nazionale Tumori—IRCSS—Fondazione ‘G. Pascale’, Naples, Italy (number 33/17 oss). Participants gave informed consent to participate in the study before taking part. All data relevant to the study are included in the article or uploaded as supplementary information. Data Availability StatementAll data relevant to the study are included in the article or uploaded as supplementary information.
- Previous studies have demonstrated that cancer exosomes express CD73.23–25 Analysis by western blotting indicated the presence of CD73 on isolated exosomes .
- CD73 was detected on exosomes isolated from healthy subjects and patients with melanoma, with no significant difference between the two groups .
- Altogether, these results suggest that changes in CD73 levels in circulating exosomes early during therapy would reflect a failure to respond to anti-PD-1 agents.
- Each sample was dispersed in deionized water and the intensity of the scattered light was measured with a detector at 90° angle at room temperature.
- We investigated the functional relevance of CD73 expressed on exosomes isolated from serum of patients with melanoma receiving nivolumab or pembrolizumab monotherapy.
The protein concentration of the purified exosomal samples was determined by Pierce BCA Protein Assay Kit . Altogether these results indicate that exosomal CD73 suppresses T-cell functions in presence of AMP by producing adenosine, via A2A adenosine receptor. In addition, the expression of the granzyme B in CD3/28 activated cells is reduced in presence of exosomes when adding the substrate AMP . A total of 10,000 events for each sample was acquired using a BD FACScalibur .
BCA-protein assay of isolated exosomal samples
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