Stephan Lang

Generation and Accumulation of Immunosuppressive Adenosine by Human CD4+CD25highFOXP3+ Regulatory T Cells

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+CD25highFOXP3+ Treg overexpress CD39 and CD73, ectonucleotidases sequentially converting ATP into AMP and adenosine, which then binds to A2a receptors on effector T cells, suppressing their functions. CD4+CD39+ and CD4+CD25high 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 A2a 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.

Myeloid‐derived suppressor cells in the peripheral blood of cancer patients contain a subset of immature neutrophils with impaired migratory properties

In tumor‐bearing mice, immunosuppressive granulocytic and monocytic MDSC have been identified. The identity and function of MDSC in cancer patients are less clear and need further characterization. We analyzed the peripheral blood of 103 patients with HNC, lung cancer, or cancers of bladder and ureter. Based on sedimentation properties in density gradients, a subset of LD‐PMN was identified and analyzed. LD‐PMN were expanded in the peripheral blood of cancer patients, suppressed proliferation, and IFN‐γ production of polyclonally stimulated T cells and thus, qualify as human MDSC. Immunophenotyping and morphological analysis revealed the accumulation of immature PMN in the MDSC fraction. Neutrophilic MDSC showed altered surface marker expression, prolonged survival, and impaired effector functions when compared with conventional, mature PMN of regular density. MDSC displayed markedly reduced chemotaxis toward tumor‐conditioned medium and lacked expression of chemokine receptors CXCR1 and CXCR2, which are normally required for PMN extravasation from the bloodstream and subsequent tissue infiltration. Collectively, our data suggest the accumulation and persistence of long‐lived, immature granulocytic MDSC with T cell‐suppressive function and impaired migratory properties in the peripheral blood of cancer patients.

Neutrophils and granulocytic myeloid-derived suppressor cells: immunophenotyping, cell biology and clinical relevance in human oncology

Accumulating evidence indicates that myeloid cells are critically involved in the pathophysiology of human cancers. In contrast to the well-characterized tumor-associated macrophages, the significance of granulocytes in cancer has only recently begun to emerge. A number of studies found increased numbers of neutrophil granulocytes and granulocytic myeloid-derived suppressor cells (GrMDSCs) both in the peripheral blood and in the tumor tissues of patients with different types of cancer. Most importantly, granulocytes have been linked to poor clinical outcome in cancer patients which suggests that these cells might have important tumor-promoting effects. In this review, we will address in detail the following major topics: (1) neutrophils and GrMDSCs in the peripheral blood of cancer patients—phenotype and functional changes; (2) neutrophils and GrMDSCs in the tumor tissue—potential mechanisms of tumor progression and (3) relevance of neutrophils and GrMDSCs for the clinical outcome of cancer patients. Furthermore, we will discuss the advantages and disadvantages of the current strategies used for identification and monitoring of human MDSCs. We propose a six-color immunophenotyping protocol that discriminates between monocytic MDSCs (MoMDSCs), two subsets of GrMDSCs and two subsets of immature myeloid cells in human cancer patients, thus, allowing for an improved characterization and understanding of these multifaceted cells.

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.

T Regulatory Type 1 Cells in Squamous Cell Carcinoma of the Head and Neck: Mechanisms of Suppression and Expansion in Advanced Disease

Purpose: Regulatory T cells play a major role in tumor escape from immunosurveillance. T regulatory cells type 1 (Tr1), a subset of regulatory T cells present in the tumor and peripheral circulation of patients with head and neck squamous cell carcinoma (HNSCC), mediate immune suppression and might contribute to tumor progression. Experimental Design: CD4+CD25−T cells were isolated from peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocytes (TIL) of 26 HNSCC patients and 10 normal controls. The Tr1 cell phenotype was determined before and after culture in the presence of interleukin (IL)-2, IL-10, and IL-15, each at 10 to 20 IU/mL. Suppression was measured in carboxyfluorescein diacetate succinimidyl ester–based proliferation assays with or without neutralizing anti-IL-10 or anti–transforming growth factor-β1 (TGF-β1) monoclonal antibodies in Transwell systems. ELISA was used to define the Tr1 cytokine profile. Results: Tr1 cells originate from CD4+CD25− precursors present in TIL and PBMC of HNSCC patients. Cytokine-driven ex vivo expansion of Tr1 precursors yielded CD4+CD25−Foxp3lowCD132+IL-10+TGF-β1+ populations that mediated higher suppression than Tr1 cells of normal controls (P < 0.0001). Tr1 cells suppressed proliferation of autologous responders via IL-10 and TGF-β1 secretion. Expression of these cytokines was higher in TIL-derived than PBMC-derived Tr1 cells (P < 0.0001). The Tr1 cell frequency and suppressor function were significantly higher in patients presenting with advanced than early disease stages and in patients “cured” by oncologic therapies than in those with active disease. Conclusions: In HNSCC, Tr1 cell generation is promoted at the tumor site. Tr1 cells use TGF-β and IL-10 to mediate suppression. They expand during disease progression and also following cancer therapy in patients with no evident disease.

Polymorphonuclear granulocytes in human head and neck cancer: enhanced inflammatory activity, modulation by cancer cells and expansion in advanced disease.

The progression of epithelial cancer is associated with an intense immunological interaction between the tumor cells and immune cells of the host. However, little is known about the interaction between tumor cells and polymorphonuclear granulocytes (PMNs) in patients with head and neck squamous cell carcinoma (HNSCC). In our study, we investigated systemic PMN‐related alterations in HNSCC, the role of tumor‐infiltrating PMNs and their modulation by the tumor microenvironment. We assessed the infiltration of HNSCC tissue by PMNs (retrospectively) and systemic PMN‐related alterations in blood values (prospectively) in HNSCC patients (n = 99 and 114, respectively) and control subjects (n = 41). PMN recruitment, apoptosis and inflammatory activity were investigated in an in vitro system of peripheral blood PMNs and a human HNSCC cell line (FaDu). HNSCC tissue exhibited considerable infiltration by PMNs, and strong infiltration was associated with poorer survival in advanced disease. PMN count, neutrophil‐to‐lymphocyte ratio and serum concentrations of CXCL8 (interleukin‐8), CCL4 (MIP‐1β) and CCL5 (RANTES) were significantly higher in the peripheral blood of HNSCC patients than in that of controls. In vitro, HNSCC‐conditioned medium inhibited apoptosis of PMNs, increased chemokinesis and chemotaxis of PMNs, induced release of lactoferrin and matrix metalloproteinase 9 by PMNs and enhanced the secretion of CCL4 by PMN. Our findings demonstrate alterations in PMN biology in HNSCC patients. In vitro, tumor‐derived factors modulate cellular functions of PMNs and increase their inflammatory activity. Thus, the interaction between HNSCC and PMNs may contribute to host‐mediated changes in the tumor microenvironment.

Adenosine and Prostaglandin E2 Cooperate in the Suppression of Immune Responses Mediated by Adaptive Regulatory T Cells

Adaptive regulatory T cells (Tr1) are induced in the periphery upon encountering cognate antigens. In cancer, their frequency is increased; however, Tr1-mediated suppression mechanisms are not yet defined. Here, we evaluate the simultaneous involvement of ectonucleotidases (CD39/CD73) and cyclooxygenase 2 (COX-2) in Tr1-mediated suppression. Human Tr1 cells were generated from peripheral blood mononuclear cell-derived, sorted CD4+CD25− T cells and incubated with autologous immature dendritic cells, irradiated COX-2+ or COX-2− tumor cells, and IL-2, IL-10, and IL-15 (each at 10–15 IU/ml) for 10 days as described (Bergmann, C., Strauss, L., Zeidler, R., Lang, S., and Whiteside, T. L. (2007) Cancer Immunol. Immunother. 56, 1429–1442). Tr1 were phenotyped by multicolor flow cytometry, and suppression of proliferating responder cells was assessed in carboxyfluorescein diacetate succinimidyl ester-based assays. ATP hydrolysis was measured using a luciferase detection assay, and levels of adenosine or prostaglandin E2 (PGE2) in cell supernatants were analyzed by mass spectrometry or ELISA, respectively. Intracellular cAMP levels were measured by enzyme immunoassay. The COX-2+ tumor induced a greater number of Tr1 than COX-2− tumor (p < 0.05). Tr1 induced by COX-2+ tumor were more suppressive, hydrolyzed more exogenous ATP (p < 0.05), and produced higher levels of adenosine and PGE2 (p < 0.05) than Tr1 induced by COX-2− tumor. Inhibitors of ectonucleotidase activity, A2A and EP2 receptor antagonists, or an inhibitor of the PKA type I decreased Tr1-mediated suppression (p < 0.05), whereas rolipram, a PDE4 inhibitor, increased the intracellular cAMP level in responder cells and their susceptibility to Tr1-mediated suppression. Tr1 present in tumors or the peripheral blood of head and neck squamous cell carcinoma patients co-expressed COX-2, CD39, and CD73. A concomitant inhibition of PGE2 and adenosine via the common intracellular cAMP pathway might be a novel approach for improving results of immune therapies for cancer.

Expression of ICOS on Human Melanoma-Infiltrating CD4+CD25highFoxp3+ T Regulatory Cells: Implications and Impact on Tumor-Mediated Immune Suppression

Objective: Interaction of ICOS with its ligand (ICOSL, B7-H2) promotes T cell responses. As CD4+CD25highFoxp3+ naturally occurring T regulatory cells in melanoma patients express ICOS, we investigated the impact of ICOS on naturally occurring T regulatory cell function. Methods: Expression of ICOS and T regulatory (Treg) cell markers was determined on CD4+CD25high T cells in PBMC and tumor-infiltrating lymphocytes from melanoma patients (n = 10) and PBMC of normal controls (n = 10) by multicolor flow cytometry. Suppression mediated by sorted ICOShigh and ICOSlow Treg was assessed in CFSE-based suppression assays with autologous CD4+CD25− responder cells (RC). Transwell inserts separating Treg from RC were used to evaluate suppression mechanisms used by Treg. ICOShigh or ICOSlow Treg were coincubated with RC ± TCR and IL-2 stimulation. ICOShigh and ICOS− Treg were also expanded under conditions previously shown to induce Tr1 from RC. Results: Treg in tumor-infiltrating lymphocytes expressed ICOS (mean fluorescence intensity = 70 ± 10), while Treg in PBMC had low ICOS expression (mean fluorescence intensity = 3.5 ± 2.5, p ≤ 0.001). ICOShigh Treg up-regulated Treg markers (p ≤ 0.0016) and mediated stronger suppression (p ≤ 0.001) relative to ICOSlow Treg. ICOShigh Treg induced Tr1 cells in nonactivated RC and Th2 cells in preactivated RC. ICOShigh Treg exposed to Tr1 cytokines expressed IL-10 and suppressed RC (92 ± 12%) in contrast to ICOSlow Treg, which mediated low suppression (21 ± 15%; p ≤ 0.0028). Conclusion: ICOShigh Treg can induce diverse immune responses in RC, depending on activation signals and cytokines present. ICOShigh Treg induce Tr1 or Th2 cells depending on the activation state of RC. In a “Tr1” cytokine milieu, ICOShigh Treg transit to Tr1.

Modulation of neutrophil granulocytes in the tumor microenvironment: Mechanisms and consequences for tumor progression

Accumulating evidence indicates a critical role of myeloid cells in the pathophysiology of human cancers. In contrast to the well-characterized tumor-associated macrophages (TAMs), the significance of granulocytes in cancer has only recently begun to emerge. Increased numbers of neutrophil granulocytes have been observed both in the peripheral blood and in the tumor tissues of patients with different types of cancer. Importantly, these studies linked neutrophils to poor clinical outcome in cancer patients which suggests that these cells might have important tumor-promoting activities. Indeed, a number of functional in vitro and in vivo studies demonstrated that tumors stimulated neutrophils to promote angiogenesis and immunosuppression, as well as migration, invasion and metastasis of the tumor cells. Therefore, it became necessary to understand the mechanisms modulating the changes in the biology and functions of neutrophils in the context of the tumor microenvironment. In this review we will discuss several functions of neutrophils that might contribute to tumor progression. Furthermore, we will address in detail the cellular and molecular mechanisms that control modulation of neutrophils in the tumor microenvironment, such as recruitment to the tumor site (chemotaxis), prolonged survival and enhanced release of protumoral mediators.

Interferon-γ and Tumor Necrosis Factor-α Differentially Affect Cytokine Expression and Migration Properties of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the capacity to differentiate into different tissue cell types such as chondrocytes, osteocytes, and adipocytes. In addition, they can home to damaged, in-flamed, and malignant tissues and display immunomodulatory properties. Since tissue-derived factors might modulate these properties, we decided to explore the impact of prototypic tissue-derived inflammatory cytokines such as TNF-alpha and IFN-gamma on immunomodulatory MSCs functions. To this end, we used primary bone marrow and cord blood-derived MSCs as well as an immortalized MSC line (V54/2) as model systems. We demonstrate that under unstimulated conditions, V54/2 cells constitutively express low levels of indoleamine 2,3-dioxygenase (IDO), exert an immunosuppressive effect on activated T-lymphocyte proliferation, secrete a distinct set of cytokines, and express a wide range of chemokine receptors. Upon stimulation, the proinflammatory cytokines IFN-gamma and TNF-alpha did not inhibit suppression of T-cell proliferation, although IDO expression was up-regulated by IFN-gamma. In contrast, TNF-alpha but not IFN-gamma amplified the cytokine production of V54/2 and primary MSCs. Interestingly, IFN-gamma was superior to TNF-alpha in up-regulating expression of chemokine receptors and migration of the V54/2 cell line, while TNF-alpha was the predominant regulator of migration in primary MSCs. Altogether, our data show that properties of MSCs depend on local environmental factors. In particular, we have shown that IFN-gamma and TNF-alpha differentially regulate cytokine expression and migration of MSCs.