Effects of exosomes present in human plasma on immune cells have not been examined in detail. Immunological studies with plasma-derived exosomes require their isolation by procedures involving ultracentrifugation. These procedures were largely developed using supernatants of cultured cells. To test biologic activities of plasma-derived exosomes, methods are necessary that ensure adequate recovery of exosome fractions free of contaminating larger vesicles, cell fragments and protein/nucleic acid aggregates. Here, an optimized method for exosome isolation from human plasma/serum specimens of normal controls (NC) or cancer patients and its advantages and pitfalls are described. To remove undesirable plasma-contaminating components, ultrafiltration of differentially-centrifuged plasma/serum followed by size-exclusion chromatography prior to ultracentrifugation facilitated the removal of contaminants. Plasma or serum was equally acceptable as a source of exosomes based on the recovered protein levels (in μg protein/mL plasma) and TEM image quality. Centrifugation on sucrose density gradients led to large exosome losses. Fresh plasma was the best source of morphologically-intact exosomes, while the use of frozen/thawed plasma decreased exosome purity but not their biologic activity. Treatments of frozen plasma with DNAse, RNAse or hyaluronidase did not improve exosome purity and are not recommended. Cancer patients’ plasma consistently yielded more isolated exosomes than did NCs’ plasma. Cancer patients’ exosomes also mediated higher immune suppression as evidenced by decreased CD69 expression on responder CD4+ T effector cells. Thus, the described procedure yields biologically-active, morphologically-intact exosomes that have reasonably good purity without large protein losses and can be used for immunological, biomarker and other studies.

ObjectiveIsolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable.MethodsPlasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells.ResultsExosomes eluting in fractions #3–5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03).ConclusionsMini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications.

Purpose Head and neck cancers (HNC) often induce profound immunosuppression which contributes to disease progression and interferes with immune-based therapies. Body fluids of HNC patients are enriched in exosomes potentially engaged in negative regulation of anti-tumor immune responses. The presence and content of exosomes derived from plasma of HNC patients 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 HNC patients and 14 healthy donors. Morphology, size, numbers and protein and molecular contents of the recovered exosomes were determined. Co-culture 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 HNC patients with active disease (AD) from those with no evident disease (NED) after oncological 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 up-regulation of regulatory T cell (Treg) suppressor functions (all at p < 0.05). Exosomes of AD patients also down-regulated NKG2D expression levels in NK cells. Conclusions Exosomes in plasma of HNC patients 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.

Summary

However, in vitro-activated B cells downregulated CD73 expression, mainly produced 59-AMP, and inhibited T-cell proliferation and cytokine production. These B cells acquire the ability to restrict potentially harmful effects of activated T cells. Thus, B cells emerge as a key regulatory component of T cell-B cell interactions, and their dual regulatory activity is mediated by the products of ATP hydrolysis, 59-AMP, and ADO. (Blood. 2013;122(1):9-18)

Purpose: Exosomes isolated from the plasma of newly diagnosed acute myeloid leukemia (AML) patients have elevated protein and transforming growth factor-beta 1 (TGF-β1) contents and inhibit natural killer (NK) cell cytotoxicity (Haematologica 96, p. 1302, 2011). A potential role of exosomes in predicting responses to chemotherapy (CT) was evaluated in AML patients undergoing treatment.Experimental Design: Plasma was obtained from AML patients at diagnosis (n = 16); post-induction CT (n = 9); during consolidation CT (n = 10); in long-term remission (Lt-CR, n = 5); and from healthy volunteers (n = 7). Exosomes were isolated by size-exclusion chromatography and ultracentrifugation. The exosomal protein, soluble TGFβ-1 levels (ELISA), and the TGF-β1 profiles (western blots) were compared among patients’ cohorts. The results were correlated with the patients’ cytogenetic profile, percentage of leukemic blast, and outcome.Results: At diagnosis, protein and TGF-β1 levels were higher (p < 0.009 and p < 0.004) in AML than control exosomes. These values decreased after induction CT (p < 0.05 and p < 0.004), increased during consolidation CT (p < 0.02 and p < 0.005), and normalized in Lt-CR. While TGF-β1 and protein levels tracked one another, TGF-β1 pro-peptide, latency-associated peptide (LAP), or mature TGF-β1 differentially decorated exosomes isolated before, during, and after CT. Only TGF-β1 pro-peptide was seen in exosomes of controls or Lt-CR patients. During consolidation CT, exosomes carried TGF-β1 pro-peptide, LAP, and low levels of mature TGF-β1. NK cell co-incubation with AML exosomes carrying all three TGF-β1 forms induced down-regulation of NKG2D expression.Conclusion: Changes in exosomal protein and/or TGF-β1 content may reflect responses to CT. The exosomal profile may suggest the presence of residual disease in patients considered to have achieved complete remission.

Exosomes, small (30–150 nm) extracellular vesicles (EVs) isolated from plasma of patients with acute myeloid leukemia (AML) carry leukemia-associated antigens and multiple inhibitory molecules. Circulating exosomes can deliver suppressive cargos to immune recipient cells, inhibiting anti-tumor activities. Pre-therapy plasma of refractory/relapsed AML patients contains elevated levels of immunosuppressive exosomes which interfere with anti-leukemia functions of activated immune cells. We show that exosomes isolated from pre-therapy plasma of the AML patients receiving adoptive NK-92 cell therapy block anti-leukemia cytotoxicity of NK-92 cells and other NK-92 cell functions. NK-92 cells do not internalize AML exosomes. Instead, signaling via surface receptors expressed on NK-92 cells, AML exosomes simultaneously deliver multiple inhibitory ligands to the cognate receptors. The signals are processed downstream and activate multiple suppressive pathways in NK-92 cells. AML exosomes reprogram NK-92 cells, interfering with their anti-leukemia functions and reducing the therapeutic potential of adoptive cell transfers. Plasma-derived exosomes interfere with immune cells used for adoptive cell therapy and may limit expected therapeutic benefits of adoptive cell therapy.

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 Ca 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 Ca 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.