Arginase-1 in Plasma-Derived Exosomes as Marker of Metastasis in Patients with Head and Neck Squamous Cell Carcinoma.

Immunoregulatory Arginase-1 (Arg-1) is present in the tumor microenvironment of solid tumors. Its association to clinicopathology and its prognostic impact are inconsistent among different tumor types and biological fluids. This study evaluated Arg-1 protein levels in tumors and the circulation of patients with head and neck squamous cell carcinoma (HNSCC) in relation to clinical stage and prognosis. Tumor Arg-1 expression was monitored via immunohistochemistry while plasma Arg-1 levels via ELISA in 37 HNSCC patients. Arg-1 presence in plasma-derived exosomes was assessed using Western blots in 20 HNSCC patients. High tumor Arg-1 expression correlated with favorable clinicopathology and longer recurrence-free survival (RFS), while high plasma Arg-1 levels were associated with unfavorable clinicopathology. All patients with low tumor and high plasma Arg-1 had nodal metastases and developed recurrence. This discrepancy was attributed to the presence of Arg-1-carrying exosomes. Arg-1 was found in plasma-derived exosomes from all HNSCC patients. High exosomal Arg-1 levels were associated with positive lymph nodes and short RFS. Circulating Arg-1+ exosomes represent a mechanism of active Arg-1 export from the tumor to the periphery. Exosomes reflected biologically relevant Arg-1 levels in metastatic HNSCC and emerged as potentially more accurate biomarkers of metastatic disease and RFS than tissue or plasma Arg-1 levels.

TGFß carrying exosomes in plasma: potential biomarkers of cancer progression in patients with head and neck squamous cell carcinoma.

OBJECTIVES: Contributions of TGFß to cancer progression are well documented. However, plasma TGFß levels often do not correlate with clinicopathological data. We examine the role of TGFß carried in exosomes isolated from murine and human plasma as a contributor to disease progression in head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: The 4-nitroquinoline-1-oxide (4-NQO) mouse model was used to study changes in TGFß expression levels during oral carcinogenesis. In human HNSCC, TGFß and Smad3 protein expression levels and TGFB1 gene expression were determined. Soluble TGFß levels were evaluated by ELISA and TGFß bioassays. Exosomes were isolated from plasma using size exclusion chromatography, and TGFß content was quantified using bioassays and bioprinted microarrays. RESULTS: During 4-NQO carcinogenesis, TGFß levels in tumour tissues and in serum increased as the tumour progressed. The TGFß content of circulating exosomes also increased. In HNSCC patients, TGFß, Smad3 and TGFB1 were overexpressed in tumour tissues and correlated with increased soluble TGFß levels. Neither TGFß expression in tumours nor levels of soluble TGFß correlated with clinicopathological data or survival. Only exosome-associated TGFß reflected tumour progression and correlated with tumour size. CONCLUSIONS: Circulating TGFß+ exosomes in the plasma of patients with HNSCC emerge as potential non-invasive biomarkers of disease progression in HNSCC.

Phase I dendritic cell p53 peptide vaccine for head and neck cancer.

BACKGROUND: p53 accumulation in head and neck squamous cell carcinoma (HNSCC) cells creates a targetable tumor antigen. Adjuvant dendritic cell (DC)-based vaccination against p53 was tested in a phase I clinical trial. EXPERIMENTAL METHODS: Monocyte-derived DC from 16 patients were loaded with two modified HLA-class I p53 peptides (Arm 1), additional Th tetanus toxoid peptide (Arm 2), or additional Th wild-type (wt) p53-specific peptide (Arm 3). Vaccine DCs (vDC) were delivered to inguinal lymph nodes at three time points. vDC phenotype, circulating p53-specific T cells, and regulatory T cells (Treg) were serially monitored by flow cytometry and cytokine production by Luminex. vDC properties were compared with those of DC1 generated with an alternative maturation regimen. RESULTS: No grade II-IV adverse events were observed. Two-year disease-free survival of 88% was favorable. p53-specific T-cell frequencies were increased postvaccination in 11 of 16 patients (69%), with IFN-γ secretion detected in four of 16 patients. Treg frequencies were consistently decreased (P = 0.006) relative to prevaccination values. The phenotype and function of DC1 were improved relative to vDC. CONCLUSION: Adjuvant p53-specific vaccination of patients with HNSCC was safe and associated with promising clinical outcome, decreased Treg levels, and modest vaccine-specific immunity. HNSCC patients' DC required stronger maturation stimuli to reverse immune suppression and improve vaccine efficacy.

Effects of adjuvant chemoradiotherapy on the frequency and function of regulatory T cells in patients with head and neck cancer.

PURPOSE: Regulatory T cells (Treg) accumulate in tumor tissues and the peripheral blood of cancer patients and may persist after therapies. This cross-sectional study examines effects of adjuvant chemoradiotherapy (CRT) on Treg numbers and function in head and neck squamous cell carcinoma (HNSCC) patients. EXPERIMENTAL DESIGN: The frequency and absolute numbers of CD4(+), ATP-hydrolyzing CD4(+)CD39(+) and CD8(+) T cells, and expression levels of CD39, CD25, TGF-ß-associated LAP and GARP on Treg were measured by flow cytometry in 40 healthy donors (NC) and 71 HNSCC patients [29 untreated with active disease (AD); 22 treated with surgery; 20 treated with CRT]. All treated subjects had no evident disease (NED) at the time of phlebotomy. In an additional cohort of 40 subjects with AD (n = 15), NED (n = 10), and NC (n = 15), in vitro sensitivity of CD4(+) T-cell subsets to cisplatin and activation-induced cell death (AICD) was tested in Annexin V-binding assays. RESULTS: CRT decreased the frequency of circulating CD4(+) T cells (P < 0.002) but increased that of CD4(+)CD39(+) Treg (P ≤ 0.001) compared with untreated or surgery-only patients. Treg frequency remained elevated for >3 years. CRT increased surface expression of LAP, GARP, and CD39 on Treg. In vitro Treg were resistant to AICD or cisplatin but conventional CD4(+) T cells (Tconv) were not. CRT-induced Treg from AD or NC subjects upregulated prosurvival proteins whereas Tconv upregulated proapoptotic Bax. CONCLUSIONS: Highly suppressive, cisplatin-resistant Treg increase in frequency and persist after CRT and could be responsible for suppression of antitumor immune responses and recurrence in HNSCC.

Pathologic and gene expression features of metastatic melanomas to the brain.

BACKGROUND: The prognosis of metastatic melanomas to the brain (MBM) is variable with prolonged survival in a subset. It is unclear whether MBM differ from extracranial metastases (EcM) and primary melanomas (PrM). METHODS: To study the biology of MBM, histopathologic analysis of tumor blocks from patients' craniotomy samples and whole-genome expression profiling (WGEP) with confirmatory immunohistochemistry were performed. RESULTS: High mononuclear infiltrate and low intratumoral hemorrhage were associated with prolonged overall survival (OS). Pathway analysis of WGEP data from 29 such craniotomy tumor blocks demonstrated that several immune-related BioCarta gene sets were associated with prolonged OS. WGEP analysis of MBM in comparison with same-patient EcM and PrM showed that MBM and EcM were similar, but both differ significantly from PrM. Immunohistochemical analysis revealed that peritumoral CD3⁺ and CD8⁺ cells were associated with prolonged OS. CONCLUSIONS: MBMs are more similar to EcM compared with PrM. Immune infiltrate is a favorable prognostic factor for MBM.

IRX-2, a novel immunotherapeutic, enhances functions of human dendritic cells.

BACKGROUND: In a recent phase II clinical trial for HNSCC patients, IRX-2, a cell-derived biologic, promoted T-cell infiltration into the tumor and prolonged overall survival. Mechanisms responsible for these IRX-2-mediated effects are unknown. We hypothesized that IRX-2 enhanced tumor antigen-(TA)-specific immunity by up-regulating functions of dendritic cells (DC). METHODOLOGY/PRINCIPAL FINDINGS: Monocyte-derived DC obtained from 18 HNSCC patients and 12 healthy donors were matured using IRX-2 or a mix of TNF-α, IL-1ß and IL-6 ("conv. mix"). Multicolor flow cytometry was used to study the DC phenotype and antigen processing machinery (APM) component expression. ELISPOT and cytotoxicity assays were used to evaluate tumor-reactive cytotoxic T lymphocytes (CTL). IL-12p70 and IL-10 production by DC was measured by Luminex® and DC migration toward CCL21 was tested in transwell migration assays. IRX-2-matured DC functions were compared with those of conv. mix-matured DC. IRX-2-matured DC expressed higher levels (p<0.05) of CD11c, CD40, CCR7 as well as LMP2, TAP1, TAP2 and tapasin than conv. mix-matured DC. IRX-2-matured DC migrated significantly better towards CCL21, produced more IL-12p70 and had a higher IL12p70/IL-10 ratio than conv. mix-matured DC (p<0.05 for all). IRX-2-matured DC carried a higher density of tumor antigen-derived peptides, and CTL primed with these DC mediated higher cytotoxicity against tumor targets (p<0.05) compared to the conv. mix-matured DC. CONCLUSION: Excellent ability of IRX-2 to induce ex vivo DC maturation in HNSCC patients explains, in part, its clinical benefits and emphasizes its utility in ex vivo maturation of DC generated for therapy.

Head and neck squamous carcinomas with exophytic and endophytic type of growth have the same prognosis after surgery and adjuvant radiotherapy.

Head and neck squamous cell carcinomas (HNSCC) are characterized by exophytic or endophytic growth. We hypothesized that the growth pattern predicts outcome and associates with distinct clinical and immunological profiles. Tumors obtained from 60 HNSCC patients treated with surgery and adjuvant radiotherapy were identified as exophytic or endophytic. Recurrence-free survival (RFS) at 42 months was determined. In a subsets of 30 patients (22 exophytic and 8 endophytic) tumor stroma and parenchyma were evaluated for infiltrating CD4(+) and CD8(+) T, dendritic, myeloid and FOXP3(+) regulatory T cells (Treg) and expression of immunosuppressive cytokines by immunohistochemistry. The localization and frequency of positive cells were determined microscopically and analyzed by hierarchical clustering to distinguish exophytic versus endophytic tumors. 34/60 patients had exophytic and 26/60 endophytic tumors. No differences in clinicopathologic data, disease progression or RFS were seen between the two cohorts. Infiltrates of CD3(+)CD8(+) T cells were larger in endophytic than exophytic tumors, while FOXP3(+) Treg, TGF-ß(+), IL-10(+), Arg-1(+), CD11b(+) cells were equally prominent in both. FOXP3(+) Treg accumulated in endophytic tumor nests, while the exophytic tumor stroma was enriched in IL-10(+) cells (both at p < 0.05). Hierarchical clustering based on immunophenotyping failed to identify different clusters in these two tumor types. However, CD68(+) macrophages and FOXP3(+) Treg showed a distinct distribution. The HNSCC growth pattern did not predict RFS. Although higher numbers and differences in localization of immunosuppressive cells in endophytic versus exophytic tumors were observed, no significant relationship was established between the growth pattern and the immune profile of infiltrating lymphocytes.

CD26 expression and adenosine deaminase activity in regulatory T cells (Treg) and CD4(+) T effector cells in patients with head and neck squamous cell carcinoma.

Adenosine deaminase (ADA) is responsible for the deamination of immunosuppressive adenosine to inosine. In human T lymphocytes, ADA is associated with dipeptidyl peptidase IV (CD26). ADA expression and activity were evaluated in regulatory T cells (Treg) and CD4(+) T effector cells (Teff) of patients with head and neck squamous cell cancer (HNSCC). CD4(+)CD39(+) and CD4(+)CD39(neg) T cells were isolated by single-cell sorting from the peripheral blood of 15 HNSCC patients and 15 healthy donors (NC). CD26/ADA expression in these cells was studied by multicolor flow cytometry, confocal microscopy, RT-PCR and immunohistochemistry in tumor tissues. ADA activity was evaluated by mass spectrometry, suppression of Teff proliferation in CFSE assays and cytokine production by Luminex. CD4(+)CD39(+) Treg had low and CD4(+)CD39(neg) Teff high CD26/ADA expression and ADA activity in NC or HNSCC. The frequency and suppressor activity of CD39(+)CD26(neg) Treg were elevated in patients relative to NC (p < 0.01). However, ADA activity in patients' CD4(+)CD39(neg) Teff was decreased (p < 0.05), resulting in extracellular adenosine accumulation. Also, patients' Teff were more sensitive to inhibitory signals delivered via adenosine receptors. IL-2, IL12 and INFγ upregulated ADA expression and activity in CD4(+)CD39(neg) Teff, whereas IL-10, PGE(2) and CADO downregulated it. The differentially expressed CD26/ADA can serve as surface markers for functionally-active CD39(+)CD26(neg) Treg.

Phenotypic and functional characteristics of CD4+ CD39+ FOXP3+ and CD4+ CD39+ FOXP3neg T-cell subsets in cancer patients.

Human CD4(+) CD39(+) regulatory T (Treg) cells hydrolyze exogenous adenosine triphosphate (ATP) and participate in immunosuppressive adenosine production. They contain two T-cell subsets whose role in mediating suppression is not understood. Frequencies of both CD4(+) CD39(+) subsets were evaluated in peripheral blood lymphocytes of 57 cancer patients and in tumor infiltrating lymphocytes (TILs) of 6 patients. CD4(+) CD39(+) and CD4(+) CD39(neg) T cells isolated using immunobeads and cell sorting were cultured under various conditions. Their conversion into CD39(+) FOXP3(+) CD25(+) or CD39(+) FOX(neg) CD25(neg) cells was monitored by multiparameter flow cytometry. Hydrolysis of exogenous ATP was measured in luminescence assays. Two CD4(+) CD39(+) cell subsets differing in expression of CD25, FOXP3, CTLA-4, CD121a, PD-1, latency associated peptide (LAP), glycoprotein A repetitions predominant (GARP), and the cytokine profile accumulated with equal frequencies in the blood and tumor tissues of cancer patients. The frequency of both subsets was significantly increased in cancer. CD39 expression levels correlated with the subsets' ability to hydrolyze ATP. Conventional CD4(+) CD39(neg) T cells incubated with IL-2 + TGF-ß expanded to generate CD4(+) CD39(+) FOXP3(+) Treg cells, while CD4(+) CD39(+) FOXP3(neg) CD25(neg) subset cells stimulated via the TCR and IL-2 converted to FOXP3(+) CTLA4(+) CD25(+) TGF-ß-expressing Treg cells. Among CD4(+) CD39(+) Treg cells, the CD4(+) CD39(+) FOXP3(neg) CD25(neg) subset serves as a reservoir of cells able to convert to Treg cells upon activation by environmental signals.

Myeloid-derived suppressor cell measurements in fresh and cryopreserved blood samples.

Myeloid-derived suppressor cells (MDSC) present in the human peripheral blood, represent a heterogeneous population of cells with monocytic and granulocytic features. To provide guidelines for reliable assessments of the frequency and function of MDSC, we compared fresh vs. cryopreserved peripheral blood mononuclear cell (PBMC) samples obtained from normal controls and patients with cancer. PBMC were obtained from 4 healthy donors and 21 patients with cancer. They were stained with labeled antibodies, and the frequency of DR⁻/LIN⁻/CD11b+, DR⁻/LIN⁻/CD15+, DR⁻/LIN⁻/CD33+ and DR(-/low)/CD14+ cells was determined by flow cytometry before and after cryopreservation. CFSE-based suppressor assays were used to test inhibitory functions of MDSC. Arginase I expression and reactive oxygen species (ROS) upregulation in MDSC subsets were evaluated by flow cytometry. The DR(-/low)/CD14+ and DR⁻/LIN⁻/CD11b+ subsets of MDSC were found to be more resistant to the cryopreservation/thawing procedure compared to the DR⁻/LIN⁻/CD15+ and DR⁻/LIN⁻/CD33+ subsets. The frequency of the latter two MDSC subsets was significantly reduced after cryopreservation. All but DR⁻/LIN⁻/CD15+ cells inhibited proliferation of autologous CSFE-labeled CD4+ cells but lost suppressor activity after cryopreservation. Only DR⁻/LIN-/CD15+ cells were positive for Arginase I, but lost its expression after cryopreservation. Only fresh DR⁻/LIN⁻/CD11b+ and DR⁻/LIN⁻/CD15+ cells produced ROS after in vitro stimulation. Studies of human MDSC should be performed in fresh blood samples. If samples have to be cryopreserved, monitoring of CD11b+ and CD14+ MDSC subsets provides the most reliable results. Arginase I expression or stimulated ROS production assessed by flow cytometry are useful markers for MDSC subsets only in fresh samples.

IRX-2, a novel biologic, favors the expansion of T effector over T regulatory cells in a human tumor microenvironment model.

IRX-2, a natural cytokine biological with multiple components, has been used in preclinical and clinical studies to promote antitumor activity of T lymphocytes. To define cellular mechanisms responsible for antitumor effects of IRX-2, its ability to induce effector T cells (Teff) was examined in a model simulating the tumor microenvironment. An in vitro model containing conventional CD4(+)CD25(-) cells co-cultured with autologous immature dendritic cells, irradiated tumor cells, and cytokines was used to study differentiation and expansion of regulatory T cells (Treg) and Teff in the presence and absence of IRX-2. Phenotype, suppressor function, signaling, and cytokine production were serially measured using flow cytometry, Western blots, CFSE-based suppressor assays, and Luminex-based analyses. The presence of IRX-2 in the co-cultures promoted the induction and expansion of IFN-γ(+)Tbet(+) Teff and significantly (p < 0.01) decreased the induction of inducible IL-10(+)TGF-ß(+) Treg. The responsible mechanism involved IFN-γ-driven T cell polarization towards Teff and suppression of Treg differentiation. In an in vitro model simulating the human tumor microenvironment, IRX-2 promoted Teff expansion and antitumor activity without inducing Treg. Thus, IRX-2 could be considered as a promising component of future antitumor therapies.

Separation of human CD4+CD39+ T cells by magnetic beads reveals two phenotypically and functionally different subsets.

OBJECTIVE: The ectonucleotidase CD39 is an enzyme involved in adenosine production. Its surface expression on human regulatory T cells (Treg) allows for their flow-cytometry-based isolation from peripheral blood. To further develop and improve this method on a scale supporting translational studies, we introduced capture of CD39(+) Treg on magnetic immunobeads. METHODS: Peripheral blood mononuclear cells (PBMC) obtained from healthy donors were used for negative selection of CD4(+) T cells on AutoMACS using antibodies (Abs) specific for all lineage(+) cells. CD4(+)CD39(+) Treg were captured by biotin-conjugated anti-CD39 Abs and anti-biotin Ab-coated magnetic beads. Isolated CD4(+)CD39(+) T cells were phenotyped by flow cytometry for Treg-associated markers: CD39, CD73, FOXP3, CD25, CTLA-4, CCR4, CD45RO and CD121a or for the absence of CD127 and CD49d. CFSE-based proliferation assays and ATP hydrolysis were used to measure Treg functions. RESULTS: The purity, recovery and viability of the separated CD4(+)CD39(+) T cells were satisfactory. The isolated CD4(+)CD39(+) T cell population consisted of FOXP3(+)CD25(+) T cells which hydrolyzed exogenous ATP and suppressed autologous CD4(+) T cell proliferation and of FOXP3(neg)CD25(neg) T cells without suppressor function. The same two subsets were detectable by flow cytometry in normal PBMC, gating on CD4(+)CD39(+), CD4(+)CD127(neg), CD4(+)CD49d(neg) or CD4(+)CD25(high) Treg. CONCLUSION: CD4(+)CD39(+) Treg capture on immunobeads led to a discovery of two CD39(+) subsets. Similar to CD39(+) Treg in the peripheral blood, half of these cells are CD25(+)FOXP3(+) active suppressor cells, while the other half are CD25(neg)FOXP3(neg) and do not mediate suppression.

Triggering of Toll-like receptor 4 expressed on human head and neck squamous cell carcinoma promotes tumor development and protects the tumor from immune attack.

Toll-like receptors (TLR) expressed on inflammatory cells play a key role in host defense against pathogens, benefiting the host. TLR are also expressed on tumor cells. To evaluate the role of TLR in tumor cells, we investigated TLR4 signaling effects on human head and neck squamous cell carcinoma (HNSCC). Tumor tissues were obtained from 27 patients with laryngeal and 12 with oral cavity cancers. Normal mucosa was obtained from 10 patients with nonneoplastic disorders. Smears for bacteria were taken from all patients during surgery. TLR4 expression in tumors and HNSCC cell lines (PCI-1, PCI-13, and PCI-30) was detected by reverse transcription-PCR and immunohistochemistry. Cell growth, apoptosis, nuclear factor-kappaB (NF-kappaB) translocation, and MyD88 and IRAK-4 expression, as well as Akt phosphorylation were measured following tumor cell exposure to the TLR4 ligand lipopolysaccharide (LPS). Tumor cell sensitivity to NK-92-mediated lysis was evaluated in 4-hour (51)Cr-release assays. Cytokine levels in HNSCC supernatants were measured in Luminex-based assays. TLR4 was expressed in all tumors, HNSCC cell lines, and normal mucosa. The TLR4 expression intensity correlated with tumor grade. LPS binding to TLR4 on tumor cells enhanced proliferation, activated phosphatidylinositol 3-kinase/Akt pathway, up-regulated IRAK-4 expression, induced nuclear NF-kappaB translocation, and increased production (P<0.05) of interleukin (IL)-6, IL-8, vascular endothelial growth factor, and granulocyte macrophage colony-stimulating factor. TLR4 triggering protected tumor cells from lysis mediated by NK-92 cells. TLR4 ligation on tumor cells supports HNSCC progression.