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Natural killer (NK) cells contribute to immunosurveillance and first-line defense in the control of tumor growth and metastasis diffusion. NK-cell-derived extracellular vesicles (NKEVs) are constitutively secreted and biologically active. They reflect the protein and genetic repertoire of originating cells, and exert antitumor activity and . Cancer can compromise NK cell functions, a status potentially reflected by their extracellular vesicles. Hence, NKEVs could, on the one hand, contribute to improve cancer therapy by interacting with tumor and/or immune cells and on the other hand, sense the actual NK cell status in cancer patients. Here, we investigated the composition of healthy donors' NKEVs, including NK microvesicles and exosomes, and their interaction with uncompromised cells of the immune system. To sense the systemic NK cell status in cancer patients, we developed an immune enzymatic test (NKExoELISA) that measures plasma NK-cell-derived exosomes, captured as tsg101 CD56 nanovesicles. NKEV mass spectrometry and cytokine analysis showed the expression of NK cell markers, i.e., NKG2D and CD94, perforin, granzymes, CD40L, and other molecules involved in cytotoxicity, homing, cell adhesion, and immune activation, together with EV markers tsg101, CD81, CD63, and CD9 in both NK-derived exosomes and microvesicles. Data are available via Proteome Xchange with identifier PXD014894. Immunomodulation studies revealed that NKEVs displayed main stimulatory functions in peripheral blood mononuclear cells (PBMCs), inducing the expression of human leukocyte antigen DR isotype (HLA-DR) and costimulatory molecules on monocytes and CD25 expression on T cells, which was maintained in the presence of lipopolysaccharide (LPS) and interleukin (IL)-10/transforming growth factor beta (TGFβ), respectively. Furthermore, NKEVs increased the CD56 NK cell fraction, suggesting that effects mediated by NKEVs might be potentially exploited in support of cancer therapy. The measurement of circulating NK exosomes in the plasma of melanoma patients and healthy donors evidenced lower levels of tsg101 CD56 exosomes in patients with respect to donors. Likewise, we detected lower frequencies of NK cells in PBMCs of these patients. These data highlight the potential of NKExoELISA to sense alterations of the NK cell immune status.

Antiangiogenics promote immune activation by reducing myeloid-derived suppressor cells (MDSCs) and enhancing natural killer (NK) and T cell functions in metastatic RCC patients. However, these effects are transient, leading to compensatory immunosuppression. Platelets (PLT) and their extracellular vesicles (PLT-EVs) modulate immune and angiogenic pathways, suggesting a role in immune reprogramming during therapy. Circulating EVs were longitudinally profiled in metastatic RCC patients (n=8) undergoing Pazopanib therapy. EVs, isolated by differential ultracentrifugation from baseline, 3- and 6-month plasma samples, were characterized by bead-based multiplex assay and nanoparticle tracking analysis. Results were correlated with blood counts, RNA-seq and flow cytometry immune profiles. Pazopanib induced temporally structured EV compartment alterations. After three months, EVs were enriched in immune markers (CD8, CD56, CD19, CD1c, HLA-DR), consistent with immune activation, whereas PLT-derived markers (CD41b, CD42a, CD29) were diminished. By six months, PLT-EV markers recovered, with CD62P and CD29 EVs co-expressing immunoregulatory and angiogenic molecules (CD209, CD105). PLT-EV abundance correlated with the expansion of regulatory T cells (Tregs), PD-L1 monocytes and MDSCs, together with suppression of NK cells. PLT activation and PDGF signaling pathways decreased in PBMC from patients with clinical benefit. Despite the small sample size and absence of functional experiments, our results suggest that Pazopanib promotes cytotoxic immune programs but, by six months, reprograms PLT-EVs towards different adhesion characteristics contributing to Treg and MDSC expansion while suppressing NK activity. PLT-EVs may influence the balance between immune activation and suppression during anti-angiogenic therapy, suggesting PLT-EVs as biomarkers and therapeutic targets in mRCC.

BackgroundColorectal cancer (CRC) remains a significant healthcare burden worldwide, characterized by a complex interplay between obesity and chronic inflammation. While the relationship between CRC, obesity and altered lipid metabolism is not fully understood, there are evidences suggesting a link between them. In this study, we hypothesized that dysregulated lipid metabolism contributes to local accumulation of foam cells (FC) in CRC, which in turn disrupts antitumor immunosurveillance.MethodsTumor infiltrating FC and CD8+ were quantified by digital pathology in patients affected by T2–T4 CRC with any N stage undergoing radical upfront surgery (n=65) and correlated with patients’ clinical outcomes. Multiparametric high-resolution flow cytometry analysis and bulk RNAseq of CRC tissue were conducted to evaluate the phenotype and transcriptomic program of immune cell infiltrate in relation to FC accumulation. The immunosuppressive effects of FC and mechanistic studies on FC-associated transforming growth factor-beta (TGF-β) and anti-PD-L1 inhibition were explored using an in-vitro human model of lipid-engulfed macrophages.ResultsFC (large CD68+ Bodipy+ macrophages) accumulated at the tumor margin in CRC samples. FChigh tumors exhibited reduced CD8+ T cells and increased regulatory T cells (Tregs). Functional transcriptional profiling depicted an immunosuppressed milieu characterized by reduced interferon gamma, memory CD8+ T cells, and activated macrophages mirrored by increased T-cell exhaustion and Treg enrichment. Furthermore, FChigh tumor phenotype was independent of standard clinical factors but correlated with high body mass index (BMI) and plasma saturated fatty acid levels. In CD8low tumors, the FChigh phenotype was associated with a 3-year disease-free survival rate of 8.6% compared with 28.7% of FClow (p=0.001). In-vitro studies demonstrated that FC significantly impact on CD8 proliferation in TFG-β dependent manner, while inhibition of TGF-β FC-related factors restored antitumor immunity.ConclusionsFC exert immunosuppressive activity through a TGF-β-related pathway, resulting in a CD8-excluded microenvironment and identifying immunosuppressed tumors with worse prognosis in patients with primary CRC. FC association with patient BMI and dyslipidemia might explain the link of CRC with obesity, and offers novel therapeutic and preventive perspectives in this specific clinical setting.

BackgroundMyeloid-derived suppressor cells (MDSC), a cornerstone of cancer-related immunosuppression, influence response to therapy and disease outcomes in melanoma patients. Nevertheless, their quantification is far from being integrated into routine clinical practice mostly because of the complex and still evolving phenotypic signatures applied to define the cell subsets. Here, we used a multistep downsizing process to verify whether a core of few markers could be sufficient to capture the prognostic potential of myeloid cells in peripheral blood mononuclear cells (PBMC) of metastatic melanoma patients.MethodsIn baseline frozen PBMC from a total of 143 stage IIIc to IV melanoma patients, we first assessed the relevant or redundant expression of myeloid and MDSC-related markers by flow cytometry (screening set, n=23 patients). Subsequently, we applied the identified panel to the development set samples (n=59 patients undergoing first/second-line therapy) to obtain prognostic variables associated with overall survival (OS) and progression-free survival (PFS) by machine learning adaptive index modeling. Finally, the identified score was confirmed in a validation set (n=61) and compared with standard clinical prognostic factors to assess its additive value in patient prognostication.ResultsThis selection process led to the identification of what we defined myeloid index score (MIS), which is composed by four cell subsets (CD14+, CD14+HLA-DRneg, CD14+PD-L1+ and CD15+ cells), whose frequencies above cut-offs stratified melanoma patients according to progressively worse prognosis. Patients with a MIS=0, showing no over-threshold value of MIS subsets, had the best clinical outcome, with a median survival of >33.6 months, while in patients with MIS 1→3, OS deteriorated from 10.9 to 6.8 and 6.0 months as the MIS increased (p<0.0001, c-index=0.745). MIS clustered patients into risk groups also according to PFS (p<0.0001). The inverse correlation between MIS and survival was confirmed in the validation set, was independent of the type of therapy and was not interfered by clinical prognostic factors. MIS HR was remarkably superior to that of lactate dehydrogenase, tumor burden and neutrophil-to-lymphocyte ratio.ConclusionThe MIS >0 identifies melanoma patients with a more aggressive disease, thus acting as a simple blood biomarker that can help tailoring therapeutic choices in real-life oncology.

Background The combination of immune checkpoint blockade (ICB) with standard therapies is becoming a common approach for overcoming resistance to cancer immunotherapy in most human malignancies including metastatic renal cell carcinoma (mRCC). In this regard, insights into the immunomodulatory properties of antiangiogenic agents may help designing multidrug schedules based on specific immune synergisms. Methods We used orthogonal transcriptomic and phenotyping platforms combined with functional analytic pipelines to elucidate the immunomodulatory effect of the antiangiogenic agent pazopanib in mRCC patients. Nine patients were studied longitudinally over a period of 6 months. We also analyzed transcriptional data from The Cancer Genome Atlas (TCGA) RCC cohort (N = 571) to assess the prognostic implications of our findings. The effect of pazopanib was assessed in vitro on NK cells and T cells. Additionally, myeloid‐derived suppressor (MDSC)‐like cells were generated from CD14+ monocytes transfected with mimics of miRNAs associated with MDSC function in the presence or absence of pazopanib. Results Pazopanib administration caused a rapid and dramatic reshaping in terms of frequency and transcriptional activity of multiple blood immune cell subsets, with a downsizing of MDSC and regulatory T cells in favor of a strong enhancement in PD‐1 expressing cytotoxic T and Natural Killer effectors. These changes were paired with an increase of the expression of transcripts reflecting activation of immune‐effector functions. This immunomodulation was marked but transient, peaking at the third month of treatment. Moreover, the intratumoral expression level of a MDSC signature (MDSC INT) was strongly associated with poor prognosis in RCC patients. In vitro experiments indicate that the observed immunomodulation might be due to an inhibitory effect on MDSC‐mediated suppression, rather than a direct effect on NK and T cells. Conclusions The marked but transient nature of this immunomodulation, peaking at the third month of treatment, provides the rationale for the use of antiangiogenics as a preconditioning strategy to improve the efficacy of ICB.  

The accrual of myeloid-derived suppressor cells (MDSCs) represents a major obstacle to effective immunotherapy in cancer patients, but the mechanisms underlying this process in the human setting remain elusive. Here, we describe a set of microRNAs (miR-146a, miR-155, miR-125b, miR-100, let-7e, miR-125a, miR-146b, miR-99b) that are associated with MDSCs and resistance to treatment with immune checkpoint inhibitors in melanoma patients. The miRs were identified by transcriptional analyses as being responsible for the conversion of monocytes into MDSCs (CD14+HLA-DRneg cells) mediated by melanoma extracellular vesicles (EVs) and were shown to recreate MDSC features upon transfection. In melanoma patients, these miRs were increased in circulating CD14+ monocytes, plasma, and tumor samples, where they correlated with the myeloid cell infiltrate. In plasma, their baseline levels clustered with the clinical efficacy of CTLA-4 or programmed cell death protein 1 (PD-1) blockade. Hence, MDSC-related miRs represent an indicator of MDSC activity in cancer patients and a potential blood marker of a poor immunotherapy outcome.

The frequency and function of regulatory T cells (Tregs) were studied in stage II–III melanoma patients who were enrolled in a phase II randomized trial of vaccination with HLA-A*0201-modified tumor peptides versus observation. The vaccinated patients received low-dose cyclophosphamide (CTX) and low-dose interleukin-2 (IL-2). Tregs were analyzed in the lymph nodes (LNs) of stage III patients who were undergoing complete lymph node dissection and in peripheral blood mononuclear cells (PBMCs) collected before vaccination and at different time points during the vaccination period. The LNs of the vaccinated patients, which were surgically removed after two rounds of vaccination and one dose of CTX, displayed a low frequency of Tregs and a less immunosuppressive environment compared with those of the untreated patients. The accurate time-course analysis of the PBMCs of patients enrolled in the vaccination arm indicated a limited and transient modulation in the frequencies of Tregs in PBMCs collected after low-dose CTX administration and a strong Treg boost in those PBMCs collected after low-dose IL-2 administration. However, a fraction of the IL-2-boosted Tregs was functionally modulated to a Th-1-like phenotype in the vaccinated patients. Moreover, low-dose IL-2 promoted the concomitant expansion of conventional activated CD4 + T cells. Despite the amplification of Tregs, IL-2 administration maintained or further increased the number of antigen-specific CD8 + T cells that were induced by vaccination as demonstrated by the ex vivo human leukocyte antigen-multimer staining and IFN-γ ELISpot assays. Our study suggests that the use of CTX as a Treg modulator should be revised in terms of the administration schedule and of patients who may benefit from this drug treatment. Despite the Treg expansion that was observed in this study, low-dose IL-2 is not detrimental to the functional activities of vaccine-primed CD8 + T cell effectors when used in the inflammatory environment of vaccination.

The aim of this study was to determine the immunogenicity and antitumor activity of autologous, tumor-derived heat shock protein gp96-peptide complex vaccine (HSPPC-96; Oncophage given with GM-CSF and IFN-alpha in pre-treated metastatic (AJCC stage IV) melanoma patients. Patients underwent surgical resection of metastatic lesions for HSPPC-96 production. HSPPC-96 was administered subcutaneously (s.c.) in four weekly intervals (first cycle). Patients with more available vaccine and absence of progressive disease received four additional injections in 2-week intervals (second cycle) or more. GM-CSF was given s.c. at the same site at days -1, 0 and +1, while IFN-alpha (3 MU) was administered s.c. at a different site at days +4 and +6. Antigen-specific anti-melanoma T and NK lymphocyte response was assessed by enzyme-linked immunospot assay on peripheral blood mononuclear cells obtained before and after vaccination. Thirty-eight patients were enrolled, 20 received at least four injections (one cycle) of HSPPC-96 and were considered assessable. Toxicity was mild and most treatment-related adverse events were local erythema and induration at the injection site. Patients receiving at least four injections of HSPPC-96 were considered evaluable for clinical response: of the 18 patients with measurable disease post surgery, 11 showed stable disease (SD). The ELISPOT assay revealed an increased class I HLA-restricted T and NK cell-mediated post-vaccination response in 5 out of 17 and 12 out of the 18 patients tested, respectively. Four of the five class I HLA-restricted T cell responses fall in the group of SD patients. Vaccination with autologous HSPPC-96 together with GM-CSF and IFN-alpha is feasible and accompanied by mild local and systemic toxicity. Both tumor-specific T cell-mediated and NK cell responses were generated in a proportion of patients. Clinical activity was limited to SD. However, both immunological and clinical responses were not improved as compared with those recorded in a previous study investigating HSPPC-96 monotherapy.

Background: Plasma EVs, a heterogeneous population of vesicles with different origins, have attracted major interest as biomarker source, especially in cancer patients. Besides containing those deriving from tumour cells, the composition and phenotype of plasma EVs might reflect immune status and its modulation in relation to anti-cancer agents. Here we investigated if the EV phenotype associated with changes in routine blood tests and peripheral blood immunophenotype in metastatic renal cell cancer patients (mRCC) undergoing tyrosine kinase inhibitor (TKI) therapy. Methods: After approval by the internal ethical committee, PBMCs and plasma samples were collected from consenting patients at baseline, 3 and 6 months during therapy and stored in liquid N2 and -80°C, respectively. EVs, isolated by two-step differential centrifugation, were evaluated by flow cytometry and western blot. PBMC immunomonitoring was performed by 10-colour cytofluorimetry. Results: EVs contained in F1 (16,500 g) and F2 (118,000 g) expressed CD9 and VLA-2 and both proteins decreased in expression after 3 months TKI administration. The amount of CD9 and VLA-2 in F1 correlated significantly with a decrease of immunosuppressive CD14 +HLA-DRneg myeloid-derived suppressor cells as well as monocyte and platelet counts in samples obtained at 3 months with respect to baseline, detected by flow cytometry of PBMCs and routine blood tests. CD9 and VLA-2 in F1 EVs also correlated inversely with CD3negCD56hi16neg cells, a subset of natural killer cells. This indicates an association of circulating EV phenotype with changes occurring at peripheral blood level in RCC patients receiving TKI. Summary/Conclusion: These preliminary data suggest that plasma EVs may reflect the immune status and the immunomodulating effects occurring during cancer therapies. Additionally, they encourage the rapid development of reliable techniques for the systematic application of body fluid EVs as immune biomarkers of liquid biopsy in cancer. Funding: This work was funded by Italian Ministry of Health grant [GR2011-02351400].

The frequency and function of regulatory T cells (Tregs) were studied in stage II-III melanoma patients who were enrolled in a phase II randomized trial of vaccination with HLA-A*0201-modified tumor peptides versus observation. The vaccinated patients received low-dose cyclophosphamide (CTX) and low-dose interleukin-2 (IL-2). Tregs were analyzed in the lymph nodes (LNs) of stage III patients who were undergoing complete lymph node dissection and in peripheral blood mononuclear cells (PBMCs) collected before vaccination and at different time points during the vaccination period. The LNs of the vaccinated patients, which were surgically removed after two rounds of vaccination and one dose of CTX, displayed a low frequency of Tregs and a less immunosuppressive environment compared with those of the untreated patients. The accurate time-course analysis of the PBMCs of patients enrolled in the vaccination arm indicated a limited and transient modulation in the frequencies of Tregs in PBMCs collected after low-dose CTX administration and a strong Treg boost in those PBMCs collected after low-dose IL-2 administration. However, a fraction of the IL-2-boosted Tregs was functionally modulated to a Th-1-like phenotype in the vaccinated patients. Moreover, low-dose IL-2 promoted the concomitant expansion of conventional activated CD4 super(+) T cells. Despite the amplification of Tregs, IL-2 administration maintained or further increased the number of antigen-specific CD8 super(+) T cells that were induced by vaccination as demonstrated by the ex vivo human leukocyte antigen-multimer staining and IFN- gamma ELISpot assays. Our study suggests that the use of CTX as a Treg modulator should be revised in terms of the administration schedule and of patients who may benefit from this drug treatment. Despite the Treg expansion that was observed in this study, low-dose IL-2 is not detrimental to the functional activities of vaccine-primed CD8 super(+) T cell effectors when used in the inflammatory environment of vaccination.