Marta Szajnik

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.

Tumor-Derived Microvesicles Induce, Expand and Up-Regulate Biological Activities of Human Regulatory T Cells (Treg)

Background Tumor-derived microvesicles (TMV) or exosomes are present in body fluids of patients with cancer and might be involved in tumor progression. The frequency and suppressor functions of peripheral blood CD4+CD25highFOXP3+ Treg are higher in patients with cancer than normal controls. The hypothesis is tested that TMV contribute to induction/expansion/and activation of human Treg. Methodology/Principal Findings TMV isolated from supernatants of tumor cells but not normal cells induced the generation and enhanced expansion of human Treg. TMV also mediated conversion of CD4+CD25neg T cells into CD4+CD25highFOXP3+ Treg. Upon co-incubation with TMV, Treg showed an increased FasL, IL-10, TGF-β1, CTLA-4, granzyme B and perforin expression (p<0.05) and mediated stronger suppression of responder cell (RC) proliferation (p<0.01). Purified Treg were resistant to TMV-mediated apoptosis relative to other T cells. TMV also increased phospho-SMAD2/3 and phospho-STAT3 expression in Treg. Neutralizing Abs specific for TGF-β1 and/or IL-10 significantly inhibited TMV ability to expand Treg. Conclusions/Significance This study suggests that TMV have immunoregulatory properties. They induce Treg, promote Treg expansion, up-regulate Treg suppressor function and enhance Treg resistance to apoptosis. Interactions of TMV with Treg represent a newly-defined mechanism that might be involved in regulating peripheral tolerance by tumors and in supporting immune evasion of human cancers.

Tumor-Derived Microvesicles Promote Regulatory T Cell Expansion and Induce Apoptosis in Tumor-Reactive Activated CD8+ T Lymphocytes

Sera of patients with cancer contain membraneous microvesicles (MV) able to induce apoptosis of activated T cells by activating the Fas/Fas ligand pathway. However, the cellular origin of MV found in cancer patients’ sera varies as do their molecular and cellular profiles. To distinguish tumor-derived MV in cancer patients’ sera, we used MAGE 3/6+ present in tumors and MV. Molecular profiles of MAGE 3/6+ MV were compared in Western blots or by flow cytometry with those of MV secreted by dendritic cells or activated T cells. These profiles were found to be distinct for each cell type. Only tumor-derived MV were MAGE 3/6+ and were variably enriched in 42-kDa Fas ligand and MHC class I but not class II molecules. Effects of MV on signaling via the TCR and IL-2R and proliferation or apoptosis of activated primary T cells and T cell subsets were also assessed. Functions of activated CD8+ and CD4+ T lymphocytes were differentially modulated by tumor-derived MV. These MV inhibited signaling and proliferation of activated CD8+ but not CD4+ T cells and induced apoptosis of CD8+ T cells, including tumor-reactive, tetramer+CD8+ T cells as detected by flow cytometry for caspase activation and annexin V binding or by DNA fragmentation. Tumor-derived but not dendritic cell-derived MV induced the in vitro expansion of CD4+CD25+FOXP3+ T regulatory cells and enhanced their suppressor activity. The data suggest that tumor-derived MV induce immune suppression by promoting T regulatory cell expansion and the demise of antitumor CD8+ effector T cells, thus contributing to tumor escape.

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.

Increased frequency and suppression by regulatory T cells in patients with acute myelogenous leukemia.

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

Blast-derived microvesicles in sera from patients with acute myeloid leukemia suppress natural killer cell function via membrane-associated transforming growth factor-β1

Background Natural killer cell cytotoxicity is decreased in patients with acute myeloid leukemia in comparison to that in normal controls. Tumor-derived microvesicles present in patients’ sera exert detrimental effects on immune cells and may influence tumor progression. Design and Methods We investigated the microvesicle protein level, molecular profile and suppression of natural killer cell activity in patients with newly diagnosed acute myeloid leukemia. Results The patients’ sera contained higher levels of microvesicles compared to the levels in controls (P<0.001). Isolated microvesicles had a distinct molecular profile: in addition to conventional microvesicle markers, they contained membrane-associated transforming growth factor-β1, MICA/MICB and myeloid blasts markers, CD34, CD33 and CD117. These microvesicles decreased natural killer cell cytotoxicity (P<0.002) and down-regulated expression of NKG2D in normal natural killer cells (P<0.001). Sera from patients with acute myeloid leukemia contained elevated levels of transforming growth factor-β, and urea-mediated dissociation of microvesicles further increased the levels of this protein. Neutralizing anti-transforming growth factor-β1 antibodies inhibited microvesicle-mediated suppression of natural killer cell activity and NKG2D down-regulation. Interleukin-15 protected natural killer cells from adverse effects of tumor-derived microvesicles. Conclusions We provide evidence for the existence in acute myeloid leukemia of a novel mechanism of natural killer cell suppression mediated by tumor-derived microvesicles and for the ability of interleukin-15 to counteract this suppression.

TLR4 signaling induced by lipopolysaccharide or paclitaxel regulates tumor survival and chemoresistance in ovarian cancer.

Toll-like receptors (TLRs) expressed on immune cells trigger inflammatory responses. TLRs are also expressed on ovarian cancer (OvCa) cells, but the consequences of signaling by the TLR4/MyD88 pathway in these cells are unclear. Here, TLR4 and MyD88 expression in OvCa tissues (n=20) and cell lines (OVCAR3, SKOV3, AD10, A2780 and CP70) was evaluated by reverse transcriptase–PCR, western blots and immunohistochemistry. Cell growth, apoptosis, nuclear factor-κB (NF-κB) translocation, IRAK4 and TRIF expression and cJun phosphorylation were measured following tumor cell exposure to the TLR4 ligands, lipopolysaccharide (LPS) or paclitaxel (PTX). Culture supernatants were tested for cytokine levels. TLR4 was expressed in all tumors, tumor cell lines and normal epithelium. MyD88 was detectable in tumor tissues and in 3/5 OvCa lines but not in normal cells. In MyD88+ SCOV3 cells, LPS or PTX binding to TLR4 induced IRAK4 activation and cJun phosphorylation, activated the NF-κB pathway and promoted interleukin (IL)-8, IL-6, vascular endothelial growth factor and monocyte chemotactic protein-1 production and resistance to drug-induced apoptosis. Silencing of TLR4 in SCOV3 cells with small interference RNA resulted in phosphorylated-cJun (p-cJun) downregulation and a loss of PTX resistance. In PTX-sensitive, MyD88neg A2780 cells, TLR4 stimulation upregulated TRIF, and TLR4 silencing eliminated this effect. Thus, TLR4/MyD88 signaling supports OvCa progression and chemoresistance, promoting immune escape.

Differential Responses of Human Regulatory T Cells (Treg) and Effector T Cells to Rapamycin

Background The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4+ CD25highFoxp3+ regulatory T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-γ chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA. Methodology/Principal Findings CD4+CD25+ and CD4+CD25neg T cells were isolated from PBMC of normal controls (n = 21) using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1–100 nM) was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4+CD25high and CD4+CD25neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4+CD25neg or CD8+CD25neg T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4+CD25+ T cells in the presence of 1–100 nM RAPA (p<0.001). RAPA-expanded Treg were largely CD4+CD25highFoxp3+ cells and were resistant to apoptosis, while CD4+CD25neg T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4+CD25neg cells. Activated Treg±RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/mTOR pathway. Conclusions/Significance RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation.

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.

Interleukin-15 enhances natural killer cell cytotoxicity in patients with acute myeloid leukemia by upregulating the activating NK cell receptors.

Interleukin-15 (IL-15) has a major role in NK-cell homeostasis. Modulation of the relative frequency and expression intensity of the NK-cell receptors by IL-15 may increase NK cell-mediated cytotoxicity in cancer patients. We investigated the receptor repertoire and measured NK-cell activity in newly diagnosed AML patients and evaluated the ex vivo effects of IL-15. The expression of the activating NK cell receptors was significantly decreased in the AML patients compared to that in NK cells of healthy donors. When NK cells obtained from AML patients were cultured with IL-15, expression of the activating receptors was significantly upregulated compared to pre-culture levels. Concomitantly, cytotoxic activity of NK cells against autologous leukemic blasts increased following IL-15 stimulation. This IL-15 induced increase in activity was blocked by neutralizing antibodies specific for the NK cell activating receptors. These pre-clinical data support the future use of IL-15 for NK cell- based therapies for AML patients.