Search

PDF file - 57 KB, Results of the experimental verification of the two predictive models

PDF file - 68 KB

Purpose: Patients with cancer have an increased frequency of circulating apoptosis-sensitive CD8+CCR7neg T cells and few CD8+CCR7+ T cells versus normal controls. The functional and clinical significance of this imbalance was investigated using peripheral blood of patients with squamous cell carcinoma of the head and neck (HNSCC).Experimental Design: The frequency of circulating CD8+ T cells co-expressing CCR7, CD45RO, CD28, and Annexin V (ANXV) was evaluated in 67 patients and 57 normal controls by flow cytometry. Spearman rank correlations among immunophenotypic profiles were analyzed. Recursive partitioning classified subjects as patients or normal controls based on CD8+CCR7+ T-cell percentages. Kaplan–Meier plots estimated disease-free survival (DFS).Results: The CD8+CCR7+ T-cell frequency was low, whereas that of total CD8+CCR7neg and ANXV-binding CD8+CCR7neg T cells was higher in patients with HNSCC than in normal controls (P < 0.001–0.0001). ANXV binding correlated with the absence of CCR7 on CD8+ T cells (P < 0.001). ANXV binding was negatively correlated with the CD8+CD45ROnegCCR7+ (TN) cell frequency (P < 0.01) but positively correlated (P < 0.01) with that of CD8+CD45RO+CCR7+ (TCM) T cells and of the two CCR7neg subsets (TPM and TTD). In recursive partitioning models, the CD8+CCR7+ T-cell frequency of 31% distinguished patients from normal controls with 77% to 88% accuracy after cross-validation. In 25 patients tested before any therapy, the CD8+CCR7+ T-cell frequency of less than 28% predicted disease recurrence within 4 years of definitive therapy (P < 0.0115).Conclusion: The CD8+CCR7+ T-cell frequency in HNSCC patients' blood tested at diagnosis can discriminate them from normal controls and predicts disease recurrence. Clin Cancer Res; 19(4); 889–99. ©2012 AACR.

PDF file - 3533 KB, The presence of CD8+CCR7+ T cells in HNSCC in situ

PDF file - 1978 KB, Decreased percentages of circulating apoptosis-resistant CD8+CCR7+ T cells in HNSCC patients and NC

PDF file - 61 KB, Percentages of ANX-binding cells within the peripheral memory (TPM) and terminally differentiating (TTD)subsets in the circulation of HNSCC patients and normal controls

Supplementary Figure 2 from 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-κB (NF-κB) 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 51Cr-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-κB 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. [Cancer Res 2009;69(7):3105–13]

Supplementary Figure Legends 1-3 from 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

Supplementary Figure 3 from 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

Supplementary Figure 1 from 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

INTRODUCTION: Small extracellular vesicles (sEV) produced by tumors and called TEX mediate communication and regulate the tumor microenvironment. As a “liquid tumor biopsy” and with the ability to induce pro-tumor reprogramming, TEX offer a promising approach to monitoring cancer progression or response to therapy. AREAS COVERED: TEX isolation from body fluids and separation by immunoaffinity capture from other EVs enables TEX molecular and functional characterization in vitro and in vivo. TEX carry membrane-bound PD-L1 and a rich cargo of other proteins and nucleic acids that reflect the tumor content and activity. TEX transfer this cargo to recipient cells, activating various molecular pathways and inducing pro-tumor transcriptional changes. TEX may interfere with immune therapies, and TEX plasma levels correlate with patients’ responses to therapy. TEX induce local and systemic alterations in immune cells which may have a prognostic value. EXPERT OPINION: TEX have a special advantage as potential cancer biomarkers. Their cargo emerges as a correlate of developing or progressing malignant disease; their phenotype mimics that of the tumor; and their functional reprogramming of immune cells provides a reading of the patients’ immune status prior and post immunotherapy. Validation of TEX and T-cell-derived sEV as cancer biomarkers is an impending future task.

The eukaryotic endomembrane system plays a key role in responses of cells to stress and maintains cell homeostasis in health and disease. Its two components, exosome biogenesis and autophagy are linked through the shared molecular machinery in the endolysosomal pathway. The distinction between exosomes derived from the endosome and extracellular vesicles (EVs) produced by secretory autophagy is difficult, because of molecular crosstalk existing between the exosome and autophagy pathways. Dichotomous effects these EVs mediate in health and disease suggest they comprise vesicles derived from both pathways. The crosstalk between these pathways provides the host immune cells with context-dependent signals that might differentially drive innate and adaptive immune responses. The messages carried by autophagy-derived EVs may be distinct from messages exosomes deliver to immune cells and might induce immune responses that are qualitatively and quantitatively different. The presence of exosome biogenesis and autophagy might allow for the calibration of immune responses under various environmental stress conditions. This hypothesis is an attempt to explain why autophagy and exosome biogenesis exist as distinct albeit interactive and partially overlapping cellular pathways. By simultaneously engaging the host immune system, exosomes and autophagy maintain the balance required for cellular homeostasis under physiological and pathological conditions.

Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (OvCa). Among various mechanisms responsible for immune suppression, arginase-1 (ARG1)-carrying small extracellular vesicles (EVs) emerge as important contributors to tumor growth and tumor escape from the host immune system. Here, we report that small EVs found in the ascites and plasma of OvCa patients contain ARG1. EVs suppress proliferation of CD4+ and CD8+ T-cells in vitro and in vivo in OvCa mouse models. In mice, ARG1-containing EVs are transported to draining lymph nodes, taken up by dendritic cells and inhibit antigen-specific T-cell proliferation. Increased expression of ARG1 in mouse OvCa cells is associated with accelerated tumor progression that can be blocked by an arginase inhibitor. Altogether, our studies show that tumor cells use EVs as vehicles to carry over long distances and deliver to immune cells a metabolic checkpoint molecule – ARG1, mitigating anti-tumor immune responses., Cancer cells employ a variety of ways to escape the immune system. Here, the authors show that ovarian cancer cells produce small extracellular vescicles containing arginase 1 that are taken up by dendritic cells in the draining lymph nodes, resulting in inhibition of antigen-specific T-cell proliferation.

Peptide antigens have been administered by different approaches as 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 two vaccine approaches using the wild-type (wt) p53 vaccine.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 SC wt p53:264-272 peptide admixed with Montanide and GM-CSF. Arm B received wt p53:264-272 peptide-pulsed dendritic cells IV. Interleukin-2 (IL-2) was administered to both cohorts in alternative cycles.Nine of 13 patients (69%) in arm A and 5 of 6 patients (83%) in arm B developed an immunologic response as determined by ELISPOT and tetramer assays. The vaccine caused no serious systemic side effects. IL-2 administration resulted in grade 3 and 4 toxicities in both arms and directly induced the 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.We found that using either vaccination approach generates comparable specific immune responses against the p53 peptide with minimal toxicity. Accordingly, our findings suggest that the use of less demanding SC approach may be as effective. Furthermore, the use of low-dose SC IL-2 as an adjuvant might have interfered with the immune response. Therefore, it may not be needed in future trials.

Human CD4(+)CD25(high)FOXP3(+) T regulatory cells (Treg) can suppress responder T cell (RC) functions by various mechanisms. In co-cultures of Treg and autologous activated RC, both cell subsets up-regulate the expression of granzymes and perforin, which might contribute to Treg-mediated suppression. Here, we investigate the sensitivity and resistance of Treg and RC to granzyme/perforin-mediated death. CD4(+)CD25(neg) RC were single cell-sorted from the peripheral blood of 25 cancer patients and 15 normal controls. These RC were carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled and co-cultured with autologous CD4(+)CD25(high)FOXP3(+) Treg +/- 150 or +/-1,000 IU/mL of interleukin-2 (IL-2) to evaluate suppression of RC proliferation. In addition, survival of the cells co-cultured for 24 h and 5 days was measured using a flow-based cytotoxicity assay. Freshly isolated Treg and RC expressed granzyme A (GrA), granzyme B (GrB), and perforin. Percentages of positive cells were higher in cancer patients than controls (p0.01) and increased upon OKT3 and IL-2 stimulation. Treg, co-cultured with RC at 150 IU/mL of IL-2, no longer expressed cytotoxins and became susceptible to RC-mediated, granzyme/perforin-dependent death. However, in co-cultures with 1,000 IU/mL of IL-2, Treg became resistant to apoptosis and induced GrB-dependent, perforin-independent death of RC. When the GrB inhibitor I or GrB-specific and GrA-specific small inhibitory ribonucleic acids were used to block the granzyme pathway in Treg, RC death, and Treg-mediated suppression of RC, proliferation were significantly inhibited. Human CD4(+)CD25(high) Treg and CD4(+)CD25(neg) RC reciprocally regulate death/growth arrest by differentially utilizing the granzyme-perforin pathway depending on IL-2 concentrations.

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-kappaB (NF-kappaB) 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-kappaB 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, MyD88(neg) A2780 cells, TLR4 stimulation upregulated TRIF, and TLR4 silencing eliminated this effect. Thus, TLR4/MyD88 signaling supports OvCa progression and chemoresistance, promoting immune escape.

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.

Background 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. Methods CD8+ Jurkat cells were incubated with MAGE 3/6+, FasL+, MHC class I+ MV isolated from sera of 60 patients with HNSCC and 25 normal controls by exclusion chromatography and ultracentrifugation. Z-VAD-FITC binding to Jurkat was measured and correlated with clinical data. Results MV from patients' sera, but not from sera of normal controls, induced Jurkat cell apoptosis. Forty-five percent T cells+MV from patients with N1-N3 disease were apoptotic versus 18% T cells+MV from patients with N0 disease (p < .008). MV from patients with active disease (AD) expressed higher FasL levels than MV from patients with no evident disease (NED) or normal controls (p ≤ .01). Conclusion MAGE 3/6+, FasL+, and MHCI+ MV in sera of patients induced T-cell apoptosis, which correlated with disease activity and the presence of lymph node metastases. © 2008 Wiley Periodicals, Inc. Head Neck, 2009

International audience; IRX-2 is a cytokine-based biologic agent that has the potential to enhance antitumor immune responses. We investigated whether IRX-2 can protect T cells from tumor-induced apoptosis. Tumor-derived microvesicles (MV) expressing FasL were purified from supernatants of tumor cells and incubated with activated CD8(+) T cells. MV induced significant CD8(+) T-cell apoptosis, as evidenced by Annexin binding (64.4+/-6.4%), caspase activation (58.1+/-7.6%), a loss of mitochondrial membrane potential (82.9+/-3.9%) and DNA fragmentation. T-cell pretreatment with IRX-2 prevented apoptosis. IRX-2-mediated cytoprotection was dose and time dependent and was comparable to effects of IL-2, IL-7 or IL-15. IRX-2 prevented MV-induced downregulation of JAK3 and TCRzeta chain and induced STAT5 activation in T cells. IRX-2 prevented MV-induced Bax and Bim upregulation (P

Aim/Background: Depletion of essential (L-tryptophan) or semi-essential (L-arginine) amino acids has been shown to suppress antitumor immune responses. Arginase-1 (Arg-1) is a cytosolic enzyme catalyzing degradation of L-arginine to L-ornithine and urea. Several studies have shown the presence of abundant Arg-1 in either tumor cells or in tumor-infiltrating myeloid cells of neutrophilic or monocytic origin. This in turn, leads to downregulation of CD3-zeta levels in T cells and correlates with suppressed antitumor immunity. In patients with ovarian cancer (OvCa), exosomes released by tumor cells (TEX) are detected in body fluids including plasma or ascites and carry proteins that are expressed by OvCa cells. Here we report that OvCa cells release Arg-1 in TEX and investigate the influence of OvCa TEX-derived Arg-1 on the antitumor effector mechanisms of immune response. Methods: TEX were isolated by ultracentrifugation or exclusion chromatography and verified by Western blotting, NanoSight and electron microscopy. The presence of Arg-1 in TEX was determined by Western blotting and the Arg-1 activity was assessed using an enzymatic assay that measured conversion of arginine into urea. Immunohistochemical Arg-1 expression data in primary OvCa lesions were correlated to clinico-pathological characteristics. Effects of TEX Arg-1 on immune cells were analyzed by in vitro proliferation assay and flow cytometry. Results: OvCa ascites contained higher levels of exosomal Arg-1 as compared with the fluids obtained from benign ovarian cysts. Enzymatically active Arg-1 was detected in TEX derived from patients’ ascites as well as from ovarian cancer cell lines. High Arg-1 expression in correlated negatively with intratumoral T-cell infiltrates and CD3-zeta expression and was associated with shorter time to recurrence (TTR). In vitro, OvCa-derived Arg-1-positive TEX inhibited CD8+ and CD4+ T-cell proliferation and decreased T-cell receptor expression. We also observed that co-culture of bone-marrow-derived dendritic cells (DC) with TEX isolated from OvCa cells results in the transfer of functionally active Arg-1 that inhibits DCs-primed proliferation of OVA-antigen specific OT-I T cells. We have used TEX isolated from OvCa cells transfected with V5-tagged Arg-1 for these experiments to ensure that this is OvCa-derived, but not endogenous DC-derived arginase that suppresses T cell proliferation. All these in vitro effects were reversed by a novel Arg-1 inhibitor (OAT-1746). Conclusions: Our findings provide the first evidence for the role of Arg-1 in the formation of an immunosuppressive microenvironment in OvCa. We identify a novel mechanism of exosomal Arg-1 distribution from the tumor cells to antigen presenting cells. Inhibition of Arg-1 activity may be an attractive novel anti-cancer strategy. Funding: NCN - OPUS 6 Program 2013/11/B/NZ6/02790, NCBIR – STRATEGMED2/265503/3/NCBIR/15 Citation Format: Malgorzata Czystowska, Marta Szajnik, Kavita Ramji, Slawomir Gruca, Artur Stefanowicz, Jakub Golab, Dominika Nowis. Ovarian cancer cells release arginase-1-containing exosomes to suppress antitumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3975. doi:10.1158/1538-7445.AM2017-3975

Purpose: Patients with cancer have an increased frequency of circulating apoptosis-sensitive CD8+CCR7neg T cells and few CD8+CCR7+ T cells versus normal controls. The functional and clinical significance of this imbalance was investigated using peripheral blood of patients with squamous cell carcinoma of the head and neck (HNSCC). Experimental Design: The frequency of circulating CD8+ T cells co-expressing CCR7, CD45RO, CD28, and Annexin V (ANXV) was evaluated in 67 patients and 57 normal controls by flow cytometry. Spearman rank correlations among immunophenotypic profiles were analyzed. Recursive partitioning classified subjects as patients or normal controls based on CD8+CCR7+ T-cell percentages. Kaplan–Meier plots estimated disease-free survival (DFS). Results: The CD8+CCR7+ T-cell frequency was low, whereas that of total CD8+CCR7neg and ANXV-binding CD8+CCR7neg T cells was higher in patients with HNSCC than in normal controls (P < 0.001–0.0001). ANXV binding correlated with the absence of CCR7 on CD8+ T cells (P < 0.001). ANXV binding was negatively correlated with the CD8+CD45ROnegCCR7+ (TN) cell frequency (P < 0.01) but positively correlated (P < 0.01) with that of CD8+CD45RO+CCR7+ (TCM) T cells and of the two CCR7neg subsets (TPM and TTD). In recursive partitioning models, the CD8+CCR7+ T-cell frequency of 31% distinguished patients from normal controls with 77% to 88% accuracy after cross-validation. In 25 patients tested before any therapy, the CD8+CCR7+ T-cell frequency of less than 28% predicted disease recurrence within 4 years of definitive therapy (P < 0.0115). Conclusion: The CD8+CCR7+ T-cell frequency in HNSCC patients' blood tested at diagnosis can discriminate them from normal controls and predicts disease recurrence. Clin Cancer Res; 19(4); 889–99. ©2012 AACR.

IRX-2 is a novel immunotherapeutic containing physiologic quantities of several cytokines which protects human T lymphocytes from tumor-induced or drug-induced apoptosis. Here, we investigate the mechanisms responsible for IRX-2-mediated protection of T lymphocytes exposed to tumor-derived microvesicles (TMV).Jurkat cells or primary human T cells ± IRX-2 were co-incubated with TMV and then examined by flow cytometry or Western blots for expression of molecules regulating cell survival (FLIP, Bcl-2, Bcl-xL, Mcl-1) or death (Fas, caspase 8, caspase 9, Bax, Bid). ANX V binding, caspase activation or cytochrome c release were also measured ± cycloheximide (CHX) or ± the Akt-specific inhibitor. Jurkat cells transfected with the cFLIP gene were used to evaluate the role of cFLIP in IRX-2-mediated protection. Effects of CHX on IRX-2-mediated protection and activation of NF-κB upon the TMV/IRX-2 treatment were also measured.IRX-2 protected T cells from apoptosis by preventing Fas overexpression induced by TMV and blocking caspase 8 activation by up-regulating cFLIP. Jurkat cells overexpressing cFLIP were more resistant to TMV-induced apoptosis than the mock-transfected cells (p0.02). Signaling via the PI3K/Akt pathway, IRX-2 corrected the imbalance of pro- versus anti-apoptotic proteins induced by TMV and promoted NF-κB translocation to the nucleus. CHX abolished IRX-2-mediated protection in T cells, suggesting that IRX-2 induces de novo synthesis of one or more proteins that are required for protection.This biologic may be therapeutically useful for protection of activated T cells from tumor-induced immune suppression and death.

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

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(+)CD25(high)FOXP3(+) Treg overexpress CD39 and CD73, ectonucleotidases sequentially converting ATP into AMP and adenosine, which then binds to A(2a) receptors on effector T cells, suppressing their functions. CD4(+)CD39(+) and CD4(+)CD25(high) 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 A(2a) 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.

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.

Interleukin-15 (IL-15) has been demonstrated to play a critical role in the regulation of natural killer (NK) cells. IL-15 induces the differentiation of NK cells from hematopoietic progenitors, stimulates the expansion of peripheral NK cells, and supports their survival. We investigated the role of IL-15 as a homeostatic regulator of NK cells in 29 patients diagnosed with acute myeloid leukemia (AML) and the potential role of IL-15 in enhancing the anti-tumor activity of NK cells in AML patients. The percentage of circulating NK cells was lower (p

An elevated frequency of CD4+CD25high regulatory T cells (Treg) has been reported in the peripheral blood in patients with various solid tumors and hematologic malignancies. Although the increase in Treg seems to be a characteristic feature of most tumors, the functional role of Treg and the mechanisms of suppression, especially in patients with hematologic malignancies, have been less well defined. We investigated Treg-mediated suppression and the responsible mechanisms in thirty newly diagnosed acute myeloid leukemia (AML) patients prior to any treatment and twenty five healthy donors (NC). The percentage of circulating CD4+ CD25high Treg was higher (p

3076 Background: NK cell cytotoxicity is tightly controlled through a balance of signals from the functionally opposing activating and inhibitory receptors found on each cell. Interleukin-15 (IL-15...

Natural killer (NK) cells lyse malignant cells without prior antigen-specific priming and play a critical role in the innate immune response. A balance of signals from activating and inhibiting receptors expressed on each NK cell controls its activity. The growth, differentiation and survival of NK cells have been found to be dependent on interleukin-15 (IL-15). Using multicolor flow cytometry we investigated the receptor repertoire and also measured the NK cell activity in twenty three patients with newly diagnosed acute myeloid leukemia (AML) prior to any treatment. Further, we investigated the ex-vivo effect of IL-15 on the NK cell repertoire and NK cell cytotoxicity. The percentage of circulating NK cells was lower (p Table 1. The effect of IL-15 on the receptor expression in AML patients NK cell Activating Receptors NK cells in AML pts at diagnosis NK cells in AML pts after IL-15 stimulation p value % positive, MFI % positive, MFI NKp30 24, 2.6 84, 6.0 0.001 NKp44 4, 2.4 71, 6.7 0.001 NKp46 32, 2.6 83, 7 0.002 NKG2C 17, 2.2 58, 6.9 0.005 NKG2D 43, 2.5 87, 7.8 0.01

7054 Background: Regulatory CD4+CD25+ T cells (T reg) are critical regulators of immune tolerance. However, the functional role of T reg in human tumor immunity has been less well studied. The frequency of circulating Treg is increased in patients with solid malignancies. We evaluated the frequency, phenotype and suppressive function of T reg in patients with acute myeloid leukemia (AML). Methods: The frequency and phenotype of CD4+ CD25high T cells were investigated in the peripheral blood of fifteen newly diagnosed AML patients prior to any treatment and fifteen healthy donors by multiparameter flow cytometry. The suppressive function of Treg was evaluated using CFSE-labeled fresh autologous CD4+CD25- T cells activated with an anti-CD3 antibody. Results: The percentage of circulating CD4+ CD25high Treg was higher (p No significant financial relationships to disclose.

3011 Background: 50% of ovarian cancers carry a p53 mutation, most of which can lead to overexpression of the protein, providing a unique target for vaccine therapy. Preclinical data shows cytotoxic T-lymphocytes can be generated against wild-type peptide and can lyse tumor cells overexpressing p53. There are many methods of antigen delivery for cancer vaccines and very few comparative studies have been conducted to determine the best of these. Here we report findings for this completed study comparing two methods of vaccination. Methods: Eligible patients had Stage III, IV, or recurrent ovarian cancer and were clinically NED at study entry. Elevated CA-125 was allowed. HLA-A2.1 and overexpression of p53 were required. A wild-type epitope (264–272) with high HLA-A2.1 affinity was utilized for vaccination. Two techniques were randomly compared: subcutaneous (SQ) peptide admixed with ISA-51 and GM-CSF adjuvants or peptide-pulsed dendritic cells given intravenously (IV). Both arms received 6 million IU/m2 IL-2 SQ for ten days beginning with cycle 3. Elispot analysis was utilized to assess immunologic response, the primary endpoint. After reaching statistical significance, accrual to the more technically difficult IV arm was halted. Results: Of 21 patients enrolled, 20 have evaluable data points; 13 on the SQ arm and 7 IV. On the SQ arm, 9 of 13 (69%) patients had evidence for immunologic response. On the IV arm, 5 of 7 (71%) patients had an immunologic response. 42% on each arm experienced some grade III or IV toxicity (increased liver enzymes on the SQ arm and fatigue and lymphopenia on the IV arm), but only one patient disenrolled due to toxicity, a cardiac arrhythmia exacerbated by IL-2. Mean overall survival (to date) on the SQ arm is 70.4 months and on the IV arm is 72.9 months. Conclusions: The patients on this study were mostly heavily treated patients with short disease-free intervals, yet we demonstrated that immune responses can be generated against a wild-type protein epitope in a significant number of patients. Both vaccine approaches were similarly effective. This result should lead to a trial of early vaccination in high- risk patients to potentially delay or even prevent recurrence. No significant financial relationships to disclose.

Regulatory CD4+CD25+ T cells (T reg) are critical regulators of immune tolerance. Increasing evidence supports the existence of elevated numbers of regulatory T cells in cancer patients. Although the increase of T cells seems to be a characteristic feature in most tumors the functional role of Treg, especially in patients with hematologic malignancies, has been less well defined. Mutlicolor flow cytomerty was used to investigated the frequency and phenotype of CD4+ CD25high T cells in the peripheral blood of thirty seven newly diagnosed acute myeloid leukemia (AML) patients (prior to any treatment) and thirty one healthy donors. The suppressive function of Treg was evaluated using CFSE-labeled fresh autologous CD4+CD25− T cells activated with an anti-CD3 antibody as responders. The percentage of circulating CD4+ CD25high Treg was higher (p < 0.0001) in the AML patients (2.9 ± 0.3%, range 0.1–7%) compared to healthy donors (0.4 ± 0.02%, range 0.1–1%). Percentages of T reg expressing Foxp3, CTLA-4, CD45 RO, Fas ligand (CD95) and GITR were significantly elevated in the circulation of AML patients compared to healthy donors: Foxp3 60vs32% p

e16508 Background: TLR4, expressed by the cells of the immune system play a role in the protection of the host against pathogens. TLRs are also expressed on human cancer cells, but their role in tumor growth is unknown. The aim of this study was to correlate the presence of TLR4 and MyD88 expression with clinicopathologic outcome in patients with ovarian cancer and to analyze the consequences of signaling via the TLR4/MyD88 pathway in ovarian cancer cell lines. Methods: Tumor specimens from 41 patients with ovarian carcinoma were evaluated for TLR4 and MyD88 by immunohistochemistry and correlated with clinical and pathologic disease features. TLR4/MyD88 expression in OVCAR3, SKOV3, and A2780 was determined using RT-PCR, WB, and immunohistochemistry. NF-kB translocation to nucleus was measured by confocal microscopy. Culture supernatants were tested for levels of cytokines in Luminex-based assays. Proliferation of cancer cells was measured in the CFSE assays. Their sensitivity to paclitaxel (PLX) was measured by Annexin V binding. Western Blot analysis was used to measure activation of the PI3K/Akt, IRAK 1, IRAK 4, and TRIF. Results: In ovarian cancer patients TLR4 and MyD88 expression by the tumor was observed in 100% and 83% of tissues, respectively. The expression of MyD88 was associated with shorter progression-free survival (42 vs 31 months, p < 0.05). Ex vivo studies showed that TLR4 was expressed on OVCAR3, SKOV3, and A2780 cell lines, while A2780 did not expressed MyD88. In MyD88+ tumor cells, LPS increased proliferation (PI 17 vs 8, p < 0.05), activated NF-kB pathway and promoted cytokine production (IL-8, IL-6, RANTES, VEGF and MCP-1). LPS and PLX binding to TLR4 on MyD88+ cells induced activation of PI3K/Akt, IRAK4, and IRAK1, but decreased expression of pro-apoptotic TRIF. In contrast, in MyD88(-) cells LPS did not induce proliferation and neither LPS nor PLX induced secretion of pro-inflammatory cytokines. Further, no changes were noted in IRAK1 expression, but strong signal was observed for TRIF. TLR4+/MyD88+ tumor cells showed grater resistance to PLX. Conclusions: Our ex vivo studies elucidate the molecular mechanisms involved in TLR4/MyD88 signaling. Ligation via TLR4 leads to tumor growth, release of proinflammatory cytokines and induction of resistance to PLX-induced apoptosis. No significant financial relationships to disclose.

Abstract 2678 Poster Board II-654 Natural killer (NK) cell cytotoxicity in patients with acute myeloid leukemia (AML) is significantly decreased relative to that in normal controls (NC). However, the mechanisms responsible for low NK cell activity in AML are not known. We considered the possibility that tumor-cell-derived microvesicles (MV) mediate suppression of NK cells. MV originate from the endosomal compartment of activated normal and neoplastic cells. Evidence suggests that tumor-derived MV exert detrimental effects on cells of the immune system and may play a role in tumor progression. To determine their contribution to immune suppression in AML, MV were isolated from sera of patients newly diagnosed with AML prior to any treatment and used to evaluate MV-mediated NK cell suppression. The protein content of MV isolated using exclusion chromatography and ultracentrifugation from sera of 19 AML patients was significantly higher than that of MV isolated from sera of 25 NC (75μg±12/mL vs 1.2μg±0.4/mL, p Disclosures: No relevant conflicts of interest to declare.