Malgorzata Czystowska

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.

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.

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.

Tumor-derived microvesicles in sera of patients with head and neck cancer and their role in tumor progression.

Tumor‐derived membranous vesicles (MV) isolated from sera of the patients with squamous cell carcinomas of the head and neck (HNSCC) induce apoptosis of activated CD8+ T cells. We tested if MV molecular profile and activity correlate with disease progression.

Abstract 2414: Comparable effect of p53 peptide vaccine in adjuvant or pulsed on dendritic cells in ovarian cancer patients: A gynecologic oncology group study

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Purpose: Peptide antigens have been administered by different approaches in cancer vaccine therapy, including direct injection or pulsed onto dendritic cells; however, the optimal delivery method is still debatable. In this study, we describe the immune response elicited by the wild-type (wt) p53 vaccine using two approaches. Experimental Design: Twenty-one HLA-A2.1 patients with stage III, IV, or recurrent ovarian cancer overexpressing the p53 protein with no evidence of disease were treated in two cohorts. Arm A received subcutaneous wt p53:264-272 peptide admixed with Montanide and GM-CSF. Arm B received wt p53:264-272 peptide-pulsed dendritic cells intravenously. Interleukin-2 (IL-2) was administered in both cohorts in alternative cycles. The immunologic response was assessed by ELISPOT and tetramer assays. Results: Nine of 13 patients (69%) in arm A and 5 of 6 patients (83%) in arm B had an immunologic response. The vaccine caused no serious systemic side effects. IL-2 administration resulted in grade 3 and 4 toxicities in both arms and directly induced expansion of T regulatory cells. The median overall survival was 40.8 and 29.6 months for arm A and B, respectively; the median progression-free survival was 4.2 and. 8.7 months, respectively. Conclusion: We demonstrated that using either vaccination approach could generate comparable specific immune responses against the wt p53 peptide with minimal toxicity. Accordingly, our findings support the utilization of the less demanding subcutaneous method. Furthermore, as IL-2 may add toxicity and induce T regulatory cells, which can suppress the immune response, therefore, it may not be needed in future trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2414.