Specific blockade CD73 alters the “exhausted” phenotype of T cells in head and neck squamous cell carcinoma

The adenosine‐induced immunosuppression hampers the immune response toward tumor cells and facilitates the tumor cells to evade immunosurveillance. CD73, an ecto‐5‐nucleotidase, is the ectoenzyme dephosphorylating extracellular AMP to adenosine. Here, using immunocompetent transgenic head and neck squamous cell carcinoma (HNSCC) mouse model, immune profiling showed high expression of CD73 on CD4⁺ and CD8⁺ T cells was associated with an “exhausted” phenotype. Further, treatment with anti‐CD73 monoclonal antibody (mAb) significantly blunted the tumor growth in the mouse model, and the blockade of CD73 reversed the “exhausted” phenotype of CD4⁺ and CD8⁺ T cells through downregulation of total expression of PD‐1 and CTLA‐4 on T cells. Whereas the population of CD4⁺CD73^hi/CD8⁺CD73^hi T cells expressed higher CTLA‐4 and PD‐1 as compared to untreated controls. In addition, the human tissue microarrays showed the expression of CD73 is upregulated on tumor infiltrating immune cells in patients with primary HNSCC. Moreover, CD73 expression is an independent prognostic factor for poor outcome in our cohort of HNSCC patients. Altogether, these findings highlight the immunoregulatory role of CD73 in the development of HNSCC and we propose that CD73 may prove to be a promising immunotherapeutic target for the treatment of HNSCC.     

Invariant NKT cells are resistant to circulating CD15+ myeloid‐derived suppressor cells in patients with head and neck cancer

Myeloid‐derived suppressor cells (MDSC) are a heterogeneous population of immature and progenitor myeloid cells with an immunosuppressive role in various types of cancer, including head and neck squamous cell carcinoma (HNSCC). However, the effect on the host immune system, especially on invariant NKT (iNKT) cells with potent anti‐tumor activity, remains unclear. In this study, we investigated the effects of circulating MDSC subsets on the peripheral lymphocytes of patients with head and neck tumors. A significant accumulation of CD15⁺ granulocytic MDSC (G‐MDSC) and CD14⁺ monocytic MDSC (M‐MDSC) was demonstrated in HNSCC patients. The percentage of G‐MDSC showed an inverse correlation with the percentage of T cells in the peripheral blood. The increased G‐MDSC was significantly associated with advanced clinical stage and poor prognosis of HNSCC patients. The proliferation and viability of T cells were suppressed by CD15⁺ cells, and the suppression was reversed by adding the hydrogen peroxide scavenger catalase. However, iNKT cell activation upon α‐galactosylceramide (αGalCer) stimulation was not affected by the presence or absence of CD15⁺ G‐MDSC. These results indicate that increased G‐MDSC negatively affects peripheral T cell immunity, but not iNKT cells, in HNSCC patients, and that T cells are more sensitive to hydrogen peroxide produced by G‐MDSC than iNKT cells. Cancer immunotherapy designed to enhance the antitumor activity of iNKT cells by stimulation with αGalCer may remain effective in the presence of G‐MDSC.     

Mature dendritic cells enriched in regulatory molecules may control regulatory T cells and the prognosis of head and neck cancer

We previously reported that regulatory T (Treg) cells expressing CTLA-4 on the cell surface are abundant in head and neck squamous cell carcinoma (HNSCC). The role of expanded Treg cells in the tumor microenvironment of HNSCC remains unclear. In this study, we reveal that the tumor microenvironment of HNSCC is characterized by the high expression of genes related to Treg cells, dendritic cells (DCs), and interleukin (IL)-17-related molecules. Increased expression of IL17A, IL17F, or IL23A contributes to a favorable prognosis of HNSCC. In the tumor microenvironment of HNSCC, IL23A and IL12B are expressed in mature dendritic cells enriched in regulatory molecules (mregDCs). The mregDCs in HNSCC are a migratory and mature phenotype; their signature genes strongly correlate with Treg signature genes in HNSCC. We also observed that IL17A was highly expressed in Th17 cells and exhausted CD8⁺ T cells in HNSCC. These data suggest that mregDCs in HNSCC may contribute to the prognosis by balancing Treg cells and effector T cells that produce IL-17. Targeting mregDCs may be a novel strategy for developing new immune therapies against HNSCC.     

Selective cytokine gene expression in renal cell carcinoma tumor cells and tumor-infiltrating lymphocytes

The progression of tumors such as renal cell carcinoma (RCC), despite the presence of substantial lymphocytic infiltrates (TIL), suggests that the ability of the local immune response to control tumor growth is impaired. Cytokine gene expression was examined to further investigate the nature of this response. Initial studies were performed with frozen tumors using PCR-assisted mRNA amplification with cytokine-specific primers for interleukin 10 (IL-10), interleukin 2 (IL-2) and interferon-gamma (IFN-γ). IL-2 mRNA was not detected, despite the presence of T cells as defined by the expression of CD3γ mRNA. In contrast, mRNA for IFN-γ was expressed in 4/9 and for IL-10 in 5/9 tumors. To confirm this, 5 fresh tumor specimens were examined, and PCR demonstrated that IL-10 mRNA was detectable in 4/5 tumors from which RNA was isolated at the time of nephrectomy. In these experiments multiple cycles and dilutions were employed to semi-quantitate the expression of IL-10. To identify potential sources of this cytokine in the tumor bed, IL-10 mRNA expression in freshly isolated lymphocytes and tumor cells, TIL lines, cultured RCC and established RCC lines was examined. Our studies demonstrate that within the tumor TIL may be one source of IL-10. Lymphocyte-enriched populations from 4/5 tumors expressed IL-10 mRNA as did 4/6 freshly isolated tumor cell preparations. IL-10 gene expression was not detected, however, in tumor cells after one passage in vitro in short-term cultured RCC tumor cells (passages 2–5) or in established RCC tumor cell lines. Finally, 4/9 CD4⁺ and 2/5 CD8⁺ TIL lines expressed IL-10 mRNA either constitutively or after stimulation with anti-CD3 antibody. This finding was associated with IL-10 production in vitro. Our studies demonstrate that IL-10 mRNA is frequently present in RCC tumors and may originate from the tumor-infiltrating mononuclear cell population. © 1995 Wiley-Liss, Inc.     

T helper responses are maintained by basal-like breast cancer cells and confer to immune modulation via upregulation of PD-1 ligands

A conspicuous T cell infiltration is frequently observed in triple-negative and/or basal-like breast cancers. Since the immunological course of breast cancer is explicitly directed by helper T cells, this study aims to determine the influence of basal-like breast cancer (BLBC) cells on CD4⁺ T cell responses. Co-cultures were established with breast cancer cell lines and CD4⁺ T cells under stimulatory conditions. Helper T cell activation, proliferation, cytokine secretion, and differentiation were assessed. Protein and mRNA expression of PD-1 ligands were determined on breast cancer cell lines. Blockade assays were performed in order to determine the functional assets of PD-1 ligation. In contrast to luminal breast cancer cells, BLBC cells allowed CD4⁺ T cell activation, proliferation, and IFN-γ secretion, but only to a certain extent. A substantial population of CD25⁺CD127^low/? regulatory T (Treg) cells was also induced in BLBC co-cultures. In return, IFN-γ stimulated the upregulation of PD-L1 (B7-H1) and/or PD-L2 (B7-DC) inhibitory molecules on the basal-like cells. In prolonged periods of co-culturing, blockade of PD-1 ligands on BLBC cell lines impaired Treg differentiation, restored IL-2 secretion, and increased CD8⁺ T cell activation. In conclusion, T helper responses were maintained by BLBC cells. On the other hand, IFN-γ secreted from Th1 and other immune cells upregulated the expression of PD-1 ligands on BLBC cells and modulated the immune reactions. Our results indicate the capacity of BLBCs to adapt to IFN-γ-mediated anti-tumor immune responses and to evade immunity via upregulation of PD-1 ligands.     

Dynamic changes in immune cell profile in head and neck squamous cell carcinoma: Immunomodulatory effects of chemotherapy

Tumor cells have evolved sophisticated means of escape from the host immune system. To date, several important immunological phenomena have been revealed in peripheral blood as well as within tumors. In the present study, we first investigated the proportion and activation status of peripheral immune regulatory cells and CD8⁺ T‐cell subsets in patients with head and neck squamous cell carcinoma (HNSCC) using a multicolor flow cytometer, and then evaluated how therapy with docetaxel, cisplatin, and 5‐fluorouracil modulated the immune cell profile in peripheral blood. The proportion of naïve T cells was lower and that of effector memory T cells (T_EM) was higher in HNSCC patients than in healthy donors. Moreover, the proportions of activated T_EM cells and effector T cells (T_EFF) were dramatically increased in patients with advanced stage disease. The proportion of regulatory T cells and CD14⁺HLA‐DR^? myeloid‐derived suppressor cells was elevated in HNSCC patients. Of note, after therapy, in addition to the transient reduction in immune regulatory cells, decreases in central memory T cells and increases in T_EFF cells were observed among CD8⁺ T‐cell subsets, suggesting differentiation from central memory T cells into T_EFF cells. Our results suggested that, despite the immunosuppressive status in HNSCC patients, tumor‐specific immune responses mediated by CD8⁺ T cells might be induced and maintained. Moreover, chemotherapy can trigger not only a transient reduction in immune regulatory cells but also further activation of CD8⁺ T cells.     

Expression of proinflammatory and proangiogenic cytokines in patients with head and neck cancer.

Altered immune, inflammatory, and angiogenesis responses are observed in patients with head and neck squamous cell carcinoma (HNSCC), and many of these responses have been linked with aggressive malignant behavior and a decrease in prognosis. In this study, we examined the hypothesis that HNSCC cells produce cytokines that regulate immune, inflammatory, and angiogenesis responses. We identified important regulatory cytokines in supernatants of well-defined and freshly cultured HNSCC cell lines by ELISA and determined whether these cytokines are detected in tumor cell lines and tissue specimens by immunohistochemistry. The serum concentration of the cytokines and cytokine-dependent acute phase inflammatory responses (i.e., fibrinogen, C-reactive protein, and erythrocyte sedimentation rate) from patients with HNSCC was determined, and the potential relationship of serum cytokine levels to tumor volume was analyzed. Cytokines interleukin (IL)-1alpha, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor were detected in similar concentration ranges in the supernatants of a panel of established University of Michigan squamous cell carcinoma (UM-SCC) cell lines and supernatants of freshly isolated primary HNSCC cultures. Evidence for the expression of IL-1alpha, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, and VEGF in HNSCC cells within tumor specimens in situ was obtained by immunohistochemistry. In a prospective comparison of the cytokine level and cytokine-inducible acute-phase proteins in serum, we report that cytokines IL-6, IL-8, and VEGF were detected at higher concentrations in the serum of patients with HNSCC compared with patients with laryngeal papilloma or age-matched control subjects (at P < 0.05). The serum concentrations of IL-8 and VEGF were found to be weakly correlated with large primary tumor volume (R2 = 0.2 and 0.4, respectively). Elevated IL-1- and IL-6-inducible acute-phase responses were also detected in cancer patients but not in patients with papilloma or control subjects (at P < 0.05). We therefore conclude that cytokines important in proinflammatory and proangiogenic responses are detectable in cell lines, tissue specimens, and serum from patients with HNSCC. These cytokines may increase the pathogenicity of HNSCC and prove useful as biomarkers or targets for therapy.     

Normal human monocytes exposed to glioma cells acquire myeloid-derived suppressor cell-like properties

Glioblastoma patients are immunosuppressed, yet glioblastomas are highly infiltrated by monocytes/macrophages. Myeloid-derived suppressor cells (MDSC; immunosuppressive myeloid cells including monocytes) have been identified in other cancers and correlate with tumor burden. We hypothesized that glioblastoma exposure causes normal monocytes to assume an MDSC-like phenotype and that MDSC are increased in glioblastoma patients. Healthy donor human CD14⁺ monocytes were cultured with human glioblastoma cell lines. Controls were cultured alone or with normal human astrocytes. After 48 hours, glioblastoma-conditioned monocytes (GCM) were purified using magnetic beads. GCM cytokine and costimulatory molecular expression, phagocytic ability, and ability to induce apoptosis in activated lymphocytes were assessed. The frequency of MDSC was assessed by flow cytometry in glioma patients'' blood and in GCM in vitro. As predicted, GCM have immunosuppressive, MDSC-like features, including reduced CD14 (but not CD11b) expression, increased immunosuppressive interleukin-10, transforming growth factor-β, and B7-H1 expression, decreased phagocytic ability, and increased ability to induce apoptosis in activated lymphocytes. Direct contact between monocytes and glioblastoma cells is necessary for complete induction of these effects. In keeping with our hypothesis, glioblastoma patients have increased circulating MDSC compared with normal donors and MDSC are increased in glioma-conditioned monocytes in vitro. To our knowledge, this has not been reported previously. Although further study is needed to directly characterize their origin and function in glioblastoma patients, these results suggest that MDSC may be an important contributor to systemic immunosuppression and can be modeled in vitro by GCM.     

NK- and T-cell subsets in malignant mesothelioma patients: Baseline pattern and changes in the context of anti-CTLA-4 therapy

Malignant mesothelioma (MM) is a highly aggressive form of cancer with limited treatment options. Although the role of NK cells has been studied in many solid tumors, the pattern of NK-cell subsets and their recognition of mesothelioma cells remain to be explored. We used RNA expression data of MM biopsies derived from the cancer genome atlas to evaluate the immune cell infiltrates. We characterized the phenotype of circulating NK and T cells of 27 MM patients before and after treatment with an anti-CTLA-4 antibody (tremelimumab). These immune cell profiles were compared to healthy controls. The RNA expression data of the MM biopsies indicated the presence of NK cells in a subgroup of patients. We demonstrated that NK cells recognize MM cell lines and that IL-15 stimulation improved NK cell-mediated lysis in vitro. Using multivariate projection models, we found that MM patients had a perturbed ratio of CD56^bright and CD56^dim NK subsets and increased serum concentrations of the cytokines IL-10, IL-8 and TNF-α. After tremelimumab treatment, the ratio between the CD56^bright and CD56^dim subsets shifted back towards physiological levels. Furthermore, the improved overall survival was correlated with low TIM-3⁺CD8⁺ T-cell frequency, high DNAM-1⁺CD56^dim NK-cell frequency and high expression levels of NKp46 on the CD56^dim NK cells before and after immune checkpoint blockade. Together, our observations suggest that NK cells infiltrate MM and that they can recognize and kill mesothelioma cells. The disease is associated with distinct lymphocytes patterns, some of which correlate with prognosis or are affected by treatment with tremelimumab.     

Radiofrequency thermal ablation of breast tumors combined with intralesional administration of IL-7 and IL-15 augments anti-tumor immune responses and inhibits tumor development and metastasis

Tumor development or recurrence is always a matter of concern following radiofrequency thermal ablation (RFA) of tumors. To determine whether combining RFA with immunologically active cytokines might induce tumor-specific immune responses against mammary carcinoma and inhibit tumor development or metastasis, we evaluated intralesional injection of IL-7 and IL-15 in RFA-treated murine tumors. We used two different breast carcinoma models: neu-overexpressing mouse mammary carcinoma (MMC) in FVBN202 transgenic mouse and 4T1 tumors in Balb/c mouse. MMC tend to relapse even in the presence of neu-specific immune responses, and 4T1 is a weakly immunogenic, aggressive and highly metastatic transplantable tumor. In vivo growth of both of these tumors is also associated with increased numbers of CD11b+Gr1+ myeloid-derived suppressor cells (MDSC). We showed for the first time that unlike RFA alone, RFA combined with the administration of intralesional IL-7 and IL-15 (after RFA), induced immune responses to tumors, inhibited tumor development and lung metastasis, and reduced MDSC. Mehran Habibi and Maciej Kmieciak have equal contribution to the work.     

Autocrine and paracrine loops between cancer cells and macrophages promote lymph node metastasis via CCR4/CCL22 in head and neck squamous cell carcinoma

Lymph node metastasis is a poor prognostic factor for patients with head and neck squamous cell carcinoma (HNSCC). However, its molecular mechanism has not yet been fully understood. In our study, we investigated the expression of CCR4 and its ligand CCL22 in the HNSCC tumor microenvironment and found that the CCR4/CCL22 axis was involved in lymph node metastasis of HNSCC. CCR4 was expressed in 20 of 31 (64.5%) human tongue cancer tissues, and its expression was significantly correlated with lymph node metastasis (p < 0.01) and lymphatic invasion (p < 0.05). CCR4 was expressed in three of five human HNSCC cell lines tested. CCR4⁺ HNSCC cells, but not CCR4^? cells, showed enhanced migration toward CCL22, indicating that functional CCR4 was expressed in HNSCC cell lines. CCL22 was also expressed in cancer cells (48.4% of tongue cancer tissues) or CD206⁺ M2‐like macrophages infiltrated in tumors and draining lymph nodes. CCL22 produced by cancer cells or CD206^high M2‐like macrophages increased the cell motility of CCR4⁺ HNSCC cells in vitro in an autocrine or paracrine manner. In the mouse SCCVII in vivo model, CCR4⁺ cancer cells, but not CCR4^? cells, metastasized to lymph nodes which contained CCL22 producing M2‐like macrophages. These results demonstrate that lymph node metastasis of CCR4⁺ HNSCC is promoted by CCL22 in an autocrine or M2‐like macrophage‐dependent paracrine manner. Therefore, the CCR4/CCL22 axis may be an attractive target for the development of diagnostic and therapeutic strategies for patients with HNSCC.     

Expansion of human T regulatory type 1 cells in the microenvironment of cyclooxygenase 2 overexpressing head and neck squamous cell carcinoma

Cyclooxygenase 2 (COX-2) overexpression and production of prostaglandin E(2) (PGE(2)) by head and neck squamous cell carcinomas (HNSCC) induce type 1 regulatory T (Tr1) cells and contribute to carcinogenesis by creating a tolerogenic milieu. To test this hypothesis, CD4(+)CD25(-) T cells obtained from the peripheral blood of 10 normal donors were cocultured with autologous dendritic cells, irradiated HNSCC cells and cytokines, interleukin 2 (IL-2), IL-10, and IL-15. HNSCC cells were either COX-2 negative, constitutively expressed COX-2, were transfected with COX-2, or had COX-2 expression knocked down by small interfering RNA. Other modifications included coculture plus or minus the COX-inhibitor, Diclofenac, or synthetic PGE(2) in the absence of HNSCC. Lymphocytes proliferating in 10-day cocultures were phenotyped by flow cytometry, studied for cytokine production by ELISA and for suppressor function in CFSE inhibition assays plus or minus anti-IL-10 or anti-transforming growth factor-beta(1) (TGF-beta(1)) monoclonal antibodies (mAb). COX-2(+) HNSCC or exogenous PGE(2) induced outgrowth of Tr1 cells with the CD3(+)CD4(+)CD25(-)IL2Rbeta(+)IL2Rgamma(+)FoxP3(+)CTLA-4(+)IL-10(+)TGF-beta(1)(+)IL-4(-) phenotype and high suppressor functions (range, 46-68%). Small interfering RNA knockout of COX-2 gene in HNSCC led to outgrowth of lymphocytes with decreased IL2Rgamma (P = 0.0001), FoxP3 (P = 0.05), and IL-10 (P = 0.035) expression and low suppressor activity (range, 26-34%). Whereas COX-2(+) cocultures contained IL-10 and TGF-beta(1) (medians, 615 and 824 pg/mL), cytokine levels were decreased (P < 0.0001) in COX-2(-) cocultures. Inhibition of COX-2 enzymatic activity in HNSCC abrogated outgrowth of Tr1 cells. Neutralizing mAbs to IL-10 and/or TGF-beta(1) abolished Tr1-mediated suppression. COX-2 overexpression in HNSCC plays a major role in the induction of Tr1 cells in the tumor microenvironment.     

Myeloid-derived suppressor cells have a central role in attenuated Listeria monocytogenes-based immunotherapy against metastatic breast cancer in young and old mice

Background:

Myeloid-derived suppressor cells (MDSCs) are present in large numbers in blood of mice and humans with cancer, and they strongly inhibit T-cell and natural killer (NK) cell responses, at young and old age. We found that a highly attenuated bacterium Listeria monocytogenes (Listeria^at)-infected MDSC and altered the immune-suppressing function of MDSC.

Methods:

Young (3 months) and old (18 months) BALB/cByJ mice with metastatic breast cancer (4T1 model) were immunised with Listeria^at semi-therapeutically (once before and twice after tumour development), and analysed for growth of metastases and primary tumour, in relation to MDSC-, CD8 T-cell and NK cell responses.

Results:

We found that Listeria^at-infected MDSC, which delivered Listeria^at predominantly to the microenvironment of metastases and primary tumours, where they spread from MDSC into tumour cells (infected tumour cells will ultimately become a target for Listeria-activated immune cells). Immunotherapy with Listeria^at significantly reduced the population of MDSC in blood and primary tumours, and converted a remaining subpopulation of MDSC into an immune-stimulating phenotype producing IL-12, in correlation with significantly improved T-cell and NK cell responses to Listeria^at at both ages. This was accompanied with a dramatic reduction in the number of metastases and tumour growth at young and old age.

Conclusions:

Although preclinical studies show that immunotherapy is less effective at old than at young age, our study demonstrates that Listeria^at-based immunotherapy can be equally effective against metastatic breast cancer at both young and old age by targeting MDSC.     

Resveratrol ameliorates Lewis lung carcinoma‐bearing mice development,decreases granulocytic myeloid‐derived suppressor cell accumulation and impairs its suppressive ability

Myeloid‐derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells which consist of 2 subsets: granulocytic MDSC (G‐MDSC) and monocytic MDSC (M‐MDSC). MDSC expand in tumor‐bearing hosts and contribute to immunotherapeutic resistance by remarkably blocking effector T‐cell activation via different mechanisms. Resveratrol (RSV) is a polyphenol and it has been widely used for its various health benefits. However, the underlying mechanism of its anti‐tumor properties remains unclear. In this study, a transplantable mouse model was used to investigate the effects of RSV on MDSC. The results showed that RSV ameliorated tumor development by decreasing G‐MDSC accumulation, impairing its suppressive ability on CD8⁺T cells and promoting M‐MDSC differentiation into CD11c⁺ and F4/80⁺ cells. Our results indicated that RSV should be considered as a modular of MDSC suppressive function and that RSV is a novel booster for tumor immunotherapy.     

Immune‐checkpoint molecules on regulatory T‐cells as a potential therapeutic target in head and neck squamous cell cancers

Immune‐checkpoint inhibitors improve the survival of head and neck squamous cell carcinoma (HNSCC) patients. Although recent studies have demonstrated that the tumor immune microenvironment (TIME) has critical roles in immunotherapy, the precise mechanisms involved are unclear. Therefore, further investigations of TIME are required for the improvement of immunotherapy. The frequency of effector regulatory T‐cells (eTregs) and the expression of immune‐checkpoint molecules (ICM) on eTregs and conventional T‐cells (Tconvs) both in peripheral blood lymphocytes (PBL) and tumor‐infiltrating lymphocytes (TIL) from HNSCC patients were analyzed by flow cytometry and their distributions were evaluated by multi‐color immunofluorescence microscopy. High frequency eTreg infiltration into HNSCC tissues was observed and high expressions of CD25, FOXP3, stimulatory‐ICM (4‐1BB, ICOS, OX40 and GITR) and inhibitory‐ICM (programmed cell death‐1 [PD‐1] and cytotoxic T‐lymphocyte‐associated protein‐4 [CTLA‐4]) were found on invasive eTregs. In contrast, the expression of stimulatory‐ICM on Tconvs was low and the expression of inhibitory‐ICM was high. In addition, ICM‐ligands (programmed cell death‐1 [PD‐L1], galectin‐9 and CEACAM‐1) were frequently expressed on cancer cells. PD‐L1 and galectin‐9 were also expressed on macrophages. PD‐1⁺ T‐cells interacted with PD‐L1⁺ cancer cells or PD‐L1⁺ macrophages. This suggested that in TIL, eTregs are highly activated, but Tconvs are exhausted or inactivated by eTregs and immune‐checkpoint systems, and ICM and eTregs are strongly involved in the creation of an immunosuppressive environment in HNSCC tissues. These suggested eTreg targeting drugs are expected to be a combination partner with immune‐checkpoint inhibitors that will improve immunotherapy of HNSCC.     

Increased recurrence and metastasis in patients whose primary head and neck squamous cell carcinomas secreted granulocyte-macrophage colony-stimulating factor and contained CD34+ natural suppressor cells

Human head and neck squamous cell carcinomas (HNSCC) that produce high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) have been shown to contain CD34⁺ natural suppressor cells that inhibit the activity of intratumoral T-cells. The present study evaluated whether GM-CSF production and the presence of CD34⁺ cells within primary HNSCC would translate into increased recurrence, metastasis or cancer-related death during the 2 years following surgical excision. Freshly excised primary HNSCC of 20 patients that subsequently developed disease, and of 17 patients that remained with no evidence of disease were analyzed for production of GM-CSF and for CD34⁺ cell content. The cancers of patients that subsequently developed recurrences or metastatic disease produced almost 4-fold the levels of GM-CSF and had approximately 2.5-fold the number of CD34⁺ cells as did cancers of patients that remained disease-free. In a second method of analysis, the prognostic significance of high vs. low GM-CSF and CD34⁺ cell values was evaluated. These analyses showed that patients whose cancers produced high GM-CSF levels or had a high CD34⁺ cell content had a disproportionately high incidence of recurrence or metastatic disease (94% and 100%, respectively), while the majority of patients whose primary cancers produced low levels of GM-CSF or had a low CD34⁺ cell content remained disease-free (16% and 19%, respectively). Our results indicate that the presence of CD34⁺ cells in GM-CSF-producing HNSCC is associated with a poorer prognosis for the cancer patients and suggest the utility of these parameters as prognostic indicators of outcome. Mechanistically, our results suggest that the presence of immune suppressive CD34⁺ cells in GM-CSF-producing HNSCC leads to increased tumor recurrence or metastasis.Int. J. Cancer 74:69–74. © 1997 Wiley-Liss, Inc.     

Tristetraprolin regulates interleukin-6, which is correlated with tumor progression in patients with head and neck squamous cell carcinoma

BACKGROUND:

Tumor‐derived cytokines play a significant role in the progression of head and neck squamous cell carcinoma (HNSCC). Targeting proteins, such as tristetraprolin (TTP), that regulate multiple inflammatory cytokines may inhibit the progression of HNSCC. However, TTP's role in cancer is poorly understood. The goal of the current study was to determine whether TTP regulates inflammatory cytokines in patients with HNSCC.

METHODS:

TTP messenger RNA (mRNA) and protein expression were determined by quantitative real‐time–polymerase chain reaction (Q‐RT‐PCR) and Western blot analysis, respectively. mRNA stability and cytokine secretion were evaluated by quantitative RT‐PCR and enzyme‐linked immunoadsorbent assay, respectively, after overexpression or knockdown of TTP in HNSCC. HNSCC tissue microarrays were immunostained for interleukin‐6 (IL‐6) and TTP.

RESULTS:

TTP expression in HNSCC cell lines was found to be inversely correlated with the secretion of IL‐6, vascular endothelial growth factor (VEGF), and prostaglandin E2 (PGE₂)_. Knockdown of TTP increased mRNA stability and the secretion of cytokines. Conversely, overexpression of TTP in HNSCC cells led to decreased secretion of IL‐6, VEGF, and PGE₂. Immunohistochemical staining of tissue microarrays for IL‐6 demonstrated that staining intensity is prognostic for poor disease‐specific survival (P = .023), tumor recurrence and development of second primary tumors (P = .014), and poor overall survival (P = .019).

CONCLUSIONS:

The results of the current study demonstrated that down‐regulation of TTP in HNSCC enhances mRNA stability and promotes secretion of IL‐6, VEGF, and PGE₂. Furthermore, high IL‐6 secretion in HNSCC tissue is a biomarker for poor prognosis. In as much as enhanced cytokine secretion is associated with poor prognosis, TTP may be a therapeutic target to reduce multiple cytokines concurrently in patients with HNSCC. Cancer 2011. © 2011 American Cancer Society.     

Involvement of local renin‐angiotensin system in immunosuppression of tumor microenvironment

To improve current cancer immunotherapies, strategies to modulate various immunosuppressive cells including myeloid derived suppressor cells (MDSC) which were shown to be negative factors in immune‐checkpoint blockade therapy, need to be developed. In the present study, we evaluated the role of the local renin‐angiotensin system (RAS) in the tumor immune‐microenvironment using murine models bearing tumor cell lines in which RAS was not involved in their proliferation and angiogenetic ability. Giving angiotensin II receptor blockers (ARB) to C57BL/6 mice bearing murine colon cancer cell line MC38 resulted in significant enhancement of tumor antigen gp70 specific T cells. ARB administration did not change the numbers of CD11b⁺ myeloid cells in tumors, but significantly reduced their T‐cell inhibitory ability along with decreased production of various immunosuppressive factors including interleukin (IL)‐6, IL‐10, vascular endothelial growth factor (VEGF), and arginase by CD11b⁺ cells in tumors. ARB also decreased expression of immunosuppressive factors such as chemokine ligand 12 and nitric oxide synthase 2 in cancer‐associated fibroblasts (CAF). Last, combination of ARB and anti‐programmed death‐ligand 1 (PD‐L1) antibodies resulted in significant augmentation of anti‐tumor effects in a CD8⁺ T cell‐dependent way. These results showed that RAS is involved in the generation of an immunosuppressive tumor microenvironment caused by myeloid cells and fibroblasts, other than the previously shown proliferative and angiogenetic properties of cancer cells and macrophages, and that ARB can transform the immunosuppressive properties of MDSC and CAF and could be used in combination with PD‐1/PD‐L1 immune‐checkpoint blockade therapy.     

Immunosuppressive activity is attenuated by Astragalus polysaccharides through remodeling the gut microenvironment in melanoma mice

Astragalus polysaccharides (APS), the main effective component of Astragalus membranaceus, can inhibit tumor growth, but the underlying mechanisms remain unclear. Previous studies have suggested that APS can regulate the gut microenvironment, including the gut microbiota and fecal metabolites. In this work, our results showed that APS could control tumor growth in melanoma-bearing mice. It could reduce the number of myeloid-derived suppressor cells (MDSC), as well as the expression of MDSC-related molecule Arg-1 and cytokines IL-10 and TGF-β, so that CD8⁺ T cells could kill tumor cells more effectively. However, while APS were administered with an antibiotic cocktail (ABX), MDSC could not be reduced, and the growth rate of tumors was accelerated. Consistent with the changes in MDSC, the serum levels of IL-6 and IL-1β were lowest in the APS group. Meanwhile, we found that fecal suspension from mice in the APS group could also reduce the number of MDSC in tumor tissues. These results revealed that APS regulated the immune function in tumor-bearing mice through remodeling the gut microbiota. Next, we focused on the results of 16S rRNA, which showed that APS significantly regulated most microorganisms, such as Bifidobacterium pseudolongum, Lactobacillus johnsonii and Lactobacillus. According to the Spearman analysis, the changes in abundance of these microorganisms were related to the increase of metabolites like glutamate and creatine, which could control tumor growth. The present study demonstrates that APS attenuate the immunosuppressive activity of MDSC in melanoma-bearing mice by remodeling the gut microbiota and fecal metabolites. Our findings reveal the therapeutic potential of APS to control tumor growth.     

Myeloid-derived suppressor cell infiltration is associated with a poor prognosis in patients with hepatocellular carcinoma

The clinicopathological features of myeloid-derived suppressor cell (MDSC) and CD8⁺ T-cell infiltration in hepatocellular carcinoma (HCC) are poorly understood. The present study examined MDSC and CD8⁺ T-cell infiltration in surgically resected primary HCC specimens and investigated the association of MDSC and CD8⁺ T-cell infiltration with clinicopathological features and patient outcomes. Using a database of 466 patients who underwent hepatic resection for HCC, immunohistochemical staining of CD33 (an MDSC marker) and CD8 was performed. High infiltration of MDSCs within the tumor was observed in patients with a poorer Barcelona Clinic Liver Cancer stage, larger tumor size, more poorly differentiated HCC, and greater presence of portal venous thrombosis, microscopic vascular thrombosis and macroscopic intrahepatic metastasis. MDSC infiltration and CD8⁺ T-cell infiltration were independent predictors of recurrence-free survival and overall survival, respectively. Stratification based on the MDSC and CD8⁺ T-cell status of the tumors was also associated with recurrence-free survival (10 year-recurrence-free survival; MDSC^highCD8⁺ T-cell^Low, 3.68%; others, 25.7%) and overall survival (10 year-overall survival; MDSC^highCD8⁺ T-cell^Low, 12.0%; others, 56.7%). In conclusion, the present large cohort study revealed that high MDSC infiltration was associated with a poor clinical outcome in patients with HCC. Furthermore, the combination of the MDSC and tumor-infiltrating CD8⁺ T-cell status enabled further classification of patients based on their outcomes.