Search

The current lack of reliable methods for quantifying extracellular vesicles (EVs) isolated from complex biofluids significantly hinders translational applications in EV research. The recently developed fluorescence nanoparticle tracking analysis (FL-NTA) allows for the detection of EV-associated proteins, enabling EV content determination. In this study, we present the first comprehensive phenotyping of bronchopulmonary lavage fluid (BALF)-derived EVs from non-small cell lung cancer (NSCLC) patients using classical EV-characterization methods as well as the FL-NTA method. We found that EV immunolabeling for the specific EV marker combined with the use of the fluorescent mode NTA analysis can provide the concentration, size, distribution, and surface phenotype of EVs in a heterogeneous solution. However, by performing FL-NTA analysis of BALF-derived EVs in comparison to plasma-derived EVs, we reveal the limitations of this method, which is suitable only for relatively pure EV isolates. For more complex fluids such as plasma, this method appears to not be sensitive enough and the measurements can be compromised. Our parallel presentation of NTA-based phenotyping of plasma and BALF EVs emphasizes the great impact of sample composition and purity on FL-NTA analysis that has to be taken into account in the further development of FL-NTA toward the detection of EV-associated cancer biomarkers.

INTRODUCTION: Small extracellular vesicles (sEV) produced by tumors and called TEX mediate communication and regulate the tumor microenvironment. As a “liquid tumor biopsy” and with the ability to induce pro-tumor reprogramming, TEX offer a promising approach to monitoring cancer progression or response to therapy. AREAS COVERED: TEX isolation from body fluids and separation by immunoaffinity capture from other EVs enables TEX molecular and functional characterization in vitro and in vivo. TEX carry membrane-bound PD-L1 and a rich cargo of other proteins and nucleic acids that reflect the tumor content and activity. TEX transfer this cargo to recipient cells, activating various molecular pathways and inducing pro-tumor transcriptional changes. TEX may interfere with immune therapies, and TEX plasma levels correlate with patients’ responses to therapy. TEX induce local and systemic alterations in immune cells which may have a prognostic value. EXPERT OPINION: TEX have a special advantage as potential cancer biomarkers. Their cargo emerges as a correlate of developing or progressing malignant disease; their phenotype mimics that of the tumor; and their functional reprogramming of immune cells provides a reading of the patients’ immune status prior and post immunotherapy. Validation of TEX and T-cell-derived sEV as cancer biomarkers is an impending future task.

Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (OvCa). Among various mechanisms responsible for immune suppression, arginase-1 (ARG1)-carrying small extracellular vesicles (EVs) emerge as important contributors to tumor growth and tumor escape from the host immune system. Here, we report that small EVs found in the ascites and plasma of OvCa patients contain ARG1. EVs suppress proliferation of CD4+ and CD8+ T-cells in vitro and in vivo in OvCa mouse models. In mice, ARG1-containing EVs are transported to draining lymph nodes, taken up by dendritic cells and inhibit antigen-specific T-cell proliferation. Increased expression of ARG1 in mouse OvCa cells is associated with accelerated tumor progression that can be blocked by an arginase inhibitor. Altogether, our studies show that tumor cells use EVs as vehicles to carry over long distances and deliver to immune cells a metabolic checkpoint molecule – ARG1, mitigating anti-tumor immune responses., Cancer cells employ a variety of ways to escape the immune system. Here, the authors show that ovarian cancer cells produce small extracellular vescicles containing arginase 1 that are taken up by dendritic cells in the draining lymph nodes, resulting in inhibition of antigen-specific T-cell proliferation.

Peptide antigens have been administered by different approaches as cancer vaccine therapy, including direct injection or pulsed onto dendritic cells; however, the optimal delivery method is still debatable. In this study, we describe the immune response elicited by two vaccine approaches using the wild-type (wt) p53 vaccine.Twenty-one HLA-A2.1 patients with stage III, IV, or recurrent ovarian cancer overexpressing the p53 protein with no evidence of disease were treated in two cohorts. Arm A received SC wt p53:264-272 peptide admixed with Montanide and GM-CSF. Arm B received wt p53:264-272 peptide-pulsed dendritic cells IV. Interleukin-2 (IL-2) was administered to both cohorts in alternative cycles.Nine of 13 patients (69%) in arm A and 5 of 6 patients (83%) in arm B developed an immunologic response as determined by ELISPOT and tetramer assays. The vaccine caused no serious systemic side effects. IL-2 administration resulted in grade 3 and 4 toxicities in both arms and directly induced the expansion of T regulatory cells. The median overall survival was 40.8 and 29.6 months for arm A and B, respectively; the median progression-free survival was 4.2 and. 8.7 months, respectively.We found that using either vaccination approach generates comparable specific immune responses against the p53 peptide with minimal toxicity. Accordingly, our findings suggest that the use of less demanding SC approach may be as effective. Furthermore, the use of low-dose SC IL-2 as an adjuvant might have interfered with the immune response. Therefore, it may not be needed in future trials.

Human CD4(+)CD25(high)FOXP3(+) T regulatory cells (Treg) can suppress responder T cell (RC) functions by various mechanisms. In co-cultures of Treg and autologous activated RC, both cell subsets up-regulate the expression of granzymes and perforin, which might contribute to Treg-mediated suppression. Here, we investigate the sensitivity and resistance of Treg and RC to granzyme/perforin-mediated death. CD4(+)CD25(neg) RC were single cell-sorted from the peripheral blood of 25 cancer patients and 15 normal controls. These RC were carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled and co-cultured with autologous CD4(+)CD25(high)FOXP3(+) Treg +/- 150 or +/-1,000 IU/mL of interleukin-2 (IL-2) to evaluate suppression of RC proliferation. In addition, survival of the cells co-cultured for 24 h and 5 days was measured using a flow-based cytotoxicity assay. Freshly isolated Treg and RC expressed granzyme A (GrA), granzyme B (GrB), and perforin. Percentages of positive cells were higher in cancer patients than controls (p0.01) and increased upon OKT3 and IL-2 stimulation. Treg, co-cultured with RC at 150 IU/mL of IL-2, no longer expressed cytotoxins and became susceptible to RC-mediated, granzyme/perforin-dependent death. However, in co-cultures with 1,000 IU/mL of IL-2, Treg became resistant to apoptosis and induced GrB-dependent, perforin-independent death of RC. When the GrB inhibitor I or GrB-specific and GrA-specific small inhibitory ribonucleic acids were used to block the granzyme pathway in Treg, RC death, and Treg-mediated suppression of RC, proliferation were significantly inhibited. Human CD4(+)CD25(high) Treg and CD4(+)CD25(neg) RC reciprocally regulate death/growth arrest by differentially utilizing the granzyme-perforin pathway depending on IL-2 concentrations.

Toll-like receptors (TLRs) expressed on immune cells trigger inflammatory responses. TLRs are also expressed on ovarian cancer (OvCa) cells, but the consequences of signaling by the TLR4/MyD88 pathway in these cells are unclear. Here, TLR4 and MyD88 expression in OvCa tissues (n=20) and cell lines (OVCAR3, SKOV3, AD10, A2780 and CP70) was evaluated by reverse transcriptase-PCR, western blots and immunohistochemistry. Cell growth, apoptosis, nuclear factor-kappaB (NF-kappaB) translocation, IRAK4 and TRIF expression and cJun phosphorylation were measured following tumor cell exposure to the TLR4 ligands, lipopolysaccharide (LPS) or paclitaxel (PTX). Culture supernatants were tested for cytokine levels. TLR4 was expressed in all tumors, tumor cell lines and normal epithelium. MyD88 was detectable in tumor tissues and in 3/5 OvCa lines but not in normal cells. In MyD88(+) SCOV3 cells, LPS or PTX binding to TLR4 induced IRAK4 activation and cJun phosphorylation, activated the NF-kappaB pathway and promoted interleukin (IL)-8, IL-6, vascular endothelial growth factor and monocyte chemotactic protein-1 production and resistance to drug-induced apoptosis. Silencing of TLR4 in SCOV3 cells with small interference RNA resulted in phosphorylated-cJun (p-cJun) downregulation and a loss of PTX resistance. In PTX-sensitive, MyD88(neg) A2780 cells, TLR4 stimulation upregulated TRIF, and TLR4 silencing eliminated this effect. Thus, TLR4/MyD88 signaling supports OvCa progression and chemoresistance, promoting immune escape.

Purpose: Regulatory CD4+CD25highFoxp3+ T cells (Treg) control peripheral immune tolerance. Patients with cancer, including those with hematologic malignancies, have elevated numbers of Treg in the peripheral circulation and in tumor tissues. However, mechanisms of suppression and clinical significance of Treg, especially in patients with acute myelogenous leukemia (AML), has not been well defined.Experimental Design: We prospectively evaluated the phenotype, function, and mechanisms of suppression used by Treg in newly diagnosed untreated AML patients. The relationship between the frequency of circulating Treg and the disease status as well as treatment outcome was also evaluated.Results: The percentage of circulating Treg was higher (P < 0.0001) and their phenotype was distinct in AML patients relative to normal controls. Suppression mediated by Treg coincubated with proliferating autologous responder cells was also higher (P < 0.001) in AML than that mediated by control Treg. Using Transwell inserts, we showed that interleukin-10 and transforming growth factor-β1 production as well as cell-to-cell contact were necessary for Treg-mediated suppression. Also, the pretreatment Treg frequency predicted response to chemotherapy. Unexpectedly, patients who achieved complete remission still had elevated frequency of Treg, which mediated high levels of suppressor activity.Conclusions: Treg accumulating in the peripheral circulation of AML patients mediate vigorous suppression via contact-dependent and contact-independent mechanisms. Patients with lower Treg frequency at diagnosis have a better response to induction chemotherapy. During the post-induction period, the Treg frequency and suppressive activity remain elevated in complete remission, suggesting that Treg are resistant to conventional chemotherapy.

Background Tumor-derived membranous vesicles (MV) isolated from sera of the patients with squamous cell carcinomas of the head and neck (HNSCC) induce apoptosis of activated CD8+ T cells. We tested if MV molecular profile and activity correlate with disease progression. Methods CD8+ Jurkat cells were incubated with MAGE 3/6+, FasL+, MHC class I+ MV isolated from sera of 60 patients with HNSCC and 25 normal controls by exclusion chromatography and ultracentrifugation. Z-VAD-FITC binding to Jurkat was measured and correlated with clinical data. Results MV from patients' sera, but not from sera of normal controls, induced Jurkat cell apoptosis. Forty-five percent T cells+MV from patients with N1-N3 disease were apoptotic versus 18% T cells+MV from patients with N0 disease (p < .008). MV from patients with active disease (AD) expressed higher FasL levels than MV from patients with no evident disease (NED) or normal controls (p ≤ .01). Conclusion MAGE 3/6+, FasL+, and MHCI+ MV in sera of patients induced T-cell apoptosis, which correlated with disease activity and the presence of lymph node metastases. © 2008 Wiley Periodicals, Inc. Head Neck, 2009

International audience; IRX-2 is a cytokine-based biologic agent that has the potential to enhance antitumor immune responses. We investigated whether IRX-2 can protect T cells from tumor-induced apoptosis. Tumor-derived microvesicles (MV) expressing FasL were purified from supernatants of tumor cells and incubated with activated CD8(+) T cells. MV induced significant CD8(+) T-cell apoptosis, as evidenced by Annexin binding (64.4+/-6.4%), caspase activation (58.1+/-7.6%), a loss of mitochondrial membrane potential (82.9+/-3.9%) and DNA fragmentation. T-cell pretreatment with IRX-2 prevented apoptosis. IRX-2-mediated cytoprotection was dose and time dependent and was comparable to effects of IL-2, IL-7 or IL-15. IRX-2 prevented MV-induced downregulation of JAK3 and TCRzeta chain and induced STAT5 activation in T cells. IRX-2 prevented MV-induced Bax and Bim upregulation (P

Purpose: Patients with cancer have an increased frequency of circulating apoptosis-sensitive CD8+CCR7neg T cells and few CD8+CCR7+ T cells versus normal controls. The functional and clinical significance of this imbalance was investigated using peripheral blood of patients with squamous cell carcinoma of the head and neck (HNSCC). Experimental Design: The frequency of circulating CD8+ T cells co-expressing CCR7, CD45RO, CD28, and Annexin V (ANXV) was evaluated in 67 patients and 57 normal controls by flow cytometry. Spearman rank correlations among immunophenotypic profiles were analyzed. Recursive partitioning classified subjects as patients or normal controls based on CD8+CCR7+ T-cell percentages. Kaplan–Meier plots estimated disease-free survival (DFS). Results: The CD8+CCR7+ T-cell frequency was low, whereas that of total CD8+CCR7neg and ANXV-binding CD8+CCR7neg T cells was higher in patients with HNSCC than in normal controls (P < 0.001–0.0001). ANXV binding correlated with the absence of CCR7 on CD8+ T cells (P < 0.001). ANXV binding was negatively correlated with the CD8+CD45ROnegCCR7+ (TN) cell frequency (P < 0.01) but positively correlated (P < 0.01) with that of CD8+CD45RO+CCR7+ (TCM) T cells and of the two CCR7neg subsets (TPM and TTD). In recursive partitioning models, the CD8+CCR7+ T-cell frequency of 31% distinguished patients from normal controls with 77% to 88% accuracy after cross-validation. In 25 patients tested before any therapy, the CD8+CCR7+ T-cell frequency of less than 28% predicted disease recurrence within 4 years of definitive therapy (P < 0.0115). Conclusion: The CD8+CCR7+ T-cell frequency in HNSCC patients' blood tested at diagnosis can discriminate them from normal controls and predicts disease recurrence. Clin Cancer Res; 19(4); 889–99. ©2012 AACR.

IRX-2 is a novel immunotherapeutic containing physiologic quantities of several cytokines which protects human T lymphocytes from tumor-induced or drug-induced apoptosis. Here, we investigate the mechanisms responsible for IRX-2-mediated protection of T lymphocytes exposed to tumor-derived microvesicles (TMV).Jurkat cells or primary human T cells ± IRX-2 were co-incubated with TMV and then examined by flow cytometry or Western blots for expression of molecules regulating cell survival (FLIP, Bcl-2, Bcl-xL, Mcl-1) or death (Fas, caspase 8, caspase 9, Bax, Bid). ANX V binding, caspase activation or cytochrome c release were also measured ± cycloheximide (CHX) or ± the Akt-specific inhibitor. Jurkat cells transfected with the cFLIP gene were used to evaluate the role of cFLIP in IRX-2-mediated protection. Effects of CHX on IRX-2-mediated protection and activation of NF-κB upon the TMV/IRX-2 treatment were also measured.IRX-2 protected T cells from apoptosis by preventing Fas overexpression induced by TMV and blocking caspase 8 activation by up-regulating cFLIP. Jurkat cells overexpressing cFLIP were more resistant to TMV-induced apoptosis than the mock-transfected cells (p0.02). Signaling via the PI3K/Akt pathway, IRX-2 corrected the imbalance of pro- versus anti-apoptotic proteins induced by TMV and promoted NF-κB translocation to the nucleus. CHX abolished IRX-2-mediated protection in T cells, suggesting that IRX-2 induces de novo synthesis of one or more proteins that are required for protection.This biologic may be therapeutically useful for protection of activated T cells from tumor-induced immune suppression and death.

Background Tumor-derived microvesicles (TMV) or exosomes are present in body fluids of patients with cancer and might be involved in tumor progression. The frequency and suppressor functions of peripheral blood CD4+CD25highFOXP3+ Treg are higher in patients with cancer than normal controls. The hypothesis is tested that TMV contribute to induction/expansion/and activation of human Treg. Methodology/Principal Findings TMV isolated from supernatants of tumor cells but not normal cells induced the generation and enhanced expansion of human Treg. TMV also mediated conversion of CD4+CD25neg T cells into CD4+CD25highFOXP3+ Treg. Upon co-incubation with TMV, Treg showed an increased FasL, IL-10, TGF-β1, CTLA-4, granzyme B and perforin expression (p

Naturally occurring regulatory T cells (nTreg) are crucial for maintaining tolerance to self and thus preventing autoimmune diseases and allograft rejections. In cancer, Treg down-regulate antitumor responses by several distinct mechanisms. This study analyzes the role the adenosinergic pathway plays in suppressive activities of human nTreg. Human CD4(+)CD25(high)FOXP3(+) Treg overexpress CD39 and CD73, ectonucleotidases sequentially converting ATP into AMP and adenosine, which then binds to A(2a) receptors on effector T cells, suppressing their functions. CD4(+)CD39(+) and CD4(+)CD25(high) T cells express low levels of adenosine deaminase (ADA), the enzyme responsible for adenosine breakdown, and of CD26, a surface-bound glycoprotein associated with ADA. In contrast, T effector cells are enriched in CD26/ADA but express low levels of CD39 and CD73. Inhibitors of ectonucleotidase activity (e.g. ARL67156) and antagonists of the A(2a) receptor (e.g. ZM241385) blocked Treg-mediated immunosuppression. The inhibition of ADA activity on effector T cells enhanced Treg-mediated immunosuppression. Thus, human nTreg characterized by the presence of CD39 and the low expression of CD26/ADA are responsible for the generation of adenosine, which plays a major role in Treg-mediated immunosuppression. The data suggest that the adenosinergic pathway represents a potential therapeutic target for regulation of immunosuppression in a broad variety of human diseases.

Background: The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4 + CD25 high Foxp3 + regulatory T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-c chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA. Methodology/Principal Findings: CD4 + CD25 + and CD4 + CD25 neg T cells were isolated from PBMC of normal controls (n=21) using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1–100 nM) was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/ mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4 + CD25 high and CD4 + CD25 neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4 + CD25 neg or CD8 + CD25 neg T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4 + CD25 + T cells in the presence of 1–100 nM RAPA (p,0.001). RAPA-expanded Treg were largely CD4 + CD25 high Foxp3 + cells and were resistant to apoptosis, while CD4 + CD25 neg T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4 + CD25 neg cells. Activated Treg6RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/ mTOR pathway. Conclusions/Significance: RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation.