Immune Suppression In Cancer Research Articles

Engineering the Cancer Genome

Over the past several years, my laboratory has employed gene targeting technology to create a series of mouse models of cancer that share genetic and pathological features of the cognate diseases in humans. We have focused particularly on models of lung cancer, both adenocarcinoma and small cell lung cancer. These models have been subjected to various types of molecular characterization, including mRNA profiling and exome/whole-genome sequencing. These efforts have defined genes and pathways that promote tumor progression and metastasis, including in the case of the adenocarcinoma model down-regulation of the lineage-restricted transcription factor Nkx2.1 and other transcriptional regulators that are involved in inducing and enforcing various differentiation states that might limit tumor progression. We have also studied the involvement of subpopulations of tumor cells in this model in which the Wnt signaling pathway is active. Data from lineage tracing experiments are consistent with this population having increased tumor propagating potential. In order to functionally characterize these and other cancer genes, we have recently developed methods to mutate genes in developing tumors using the CRISPR/Cas9 system. Using tri-functional lentiviral, we have developed tumors that have undergone Cas9-mediated mutation of a series of genes of interest. Several examples of the application of this method will be reviewed. As another example of cancer genome manipulation, we have engineered tumors of the lung and other sites to express specific T cell antigens and/or NK cell ligands. These models have allowed a detailed investigation of adaptive and innate immune responses to a developing tumor. We have used the models to explore mechanisms of immune suppression in cancer and develop methods to elicit improved anti-tumor immune responses. DisclosuresNo relevant conflicts of interest to declare.

Myeloid-derived suppressor cells in B cell malignancies.

Tumor cells use several mechanisms such as soluble immune modulators or suppressive immune cells to evade from anti-tumor responses. Immunomodulatory cytokines, such as transforming growth factor-β, interleukin (IL)-10, and IL-35, soluble factors, such as adenosine, immunosuppressive cells, such as regulatory T cells, NKT cells and myeloid-derived suppressor cells (MDSCs), are the main orchestra leaders involved in immune suppression in cancer by which tumor cells can freely expand without immune cell-mediated interference. Among them, MDSCs have attracted much attention as they represent a heterogenous population derived from myeloid progenitors that are expanded in tumor condition and can also shift toward other myeloid cells, such as macrophages and dendritic cells, after tumor clearing. MDSCs exert their immunosuppressive effects through various immune and non-immune mechanisms which make them as potent tumor-promoting cells. Although, there are several studies regarding the immunobiology of MDSCs in different solid tumors, little is known about the precise characteristics of these cells in hematological malignancies, particularly B cell malignancies. In this review, we tried to clarify the precise role of MDSCs in B cell-derived malignancies.

Abstract LB-226: Depletion of kynurenine using an engineered therapeutic enzyme potently inhibits cancer immune checkpoints both as a monotherapy and in combination with anti-PD-1

Abstract Introduction: The kynurenine pathway is of key importance for immune suppression in cancer, a function exerted primarily via kynurenine as an AhR ligand that potently impairs adaptive immune responses. Inhibition of kynurenine (Kyn) synthesis, mediated by IDO1, TDO (and possibly IDO2) is of great interest for cancer immune checkpoint inhibition, with 3-4 IDO1 inhibitors currently in clinical development. However: (1) IDO1 inhibition has marginal anti-cancer effects as a monotherapy; (2) IDO1 inhibitors only block one of the two major pathways for Kynureine synthesis (the other being via TDO) and (3) IDO1 inhibition does generally not impact the serum concentration of Kynurenine and thus no pharmacodynamic marker is available. Experimental: We postulated that administration of a therapeutic enzyme (Kynureninase) that can degrade Kyn into non-toxic and immunologically inactive metabolites (Alanine and anthranilic acid) may be able to: (a) potently relieve cancer immune suppression. An extensive protein engineering campaign was carried out to develop a Kynureninase suitable for therapeutic administration which was then PEGylated (Kynase-PEG) to enable long circulation persistence. Kynase-PEG was evaluated in the well-established B16-OVA melanoma model in wild type C57BL/6J mice as a monotherapy and in combination with anti-PD-1 administration. Quantitation of tumor size/regression, histology, as well as flow cytometric analyses assessing lymphocytes in the spleen, tumor (TILs) and tumor-draining lymph nodes (dLNs) was used to evaluate efficacy. Summary: Administration of KYNase-PEG in B16-OVA melanoma allografts in C57BL/6J mice reduced serum Kyn levels and resulted in significant tumor growth retardation and extended survival in a manner indistinguishable from that observed with immune checkpoint inhibitors anti-PD1 (clone RMP1-14) or anti-CTLA-4 (clone 9D9) antibodies. KYNase-PEG administration did not display anti-tumor activity in NOD-scid Il2Rγ-/- mice or in IDO1-/- mice revealing that the function of the enzyme is dependent on adaptive immune responses and also on the function of stromal IDO1. Consistent with the hypothesis that depletion of Kyn relieves immune inhibition, we observed a marked increase in CD8+ TILs expressing Gzm B + IFNγ, enhanced proliferation of CD4+ and CD8+ T cells in the tumor dLNs as well as changes in tumor neovascularization. Importantly, combination of KYNase-PEG and anti-PD-1 administration resulted in complete tumor eradication in 60% of the animals (n = 10 per group) for >100 days, with all the surviving animals completely rejecting tumors following re-challenge. For comparison, anti-PD-1 treatment alone, only led to 20% long-term survival in this model. Conclusions: We demonstrated that unlike IDO1 inhibitors, KYNase-PEG displays a significant anti-tumor efficacy as a monotherapy as well as excellent synergism with anti-PD1. The observed increase in tumor infiltration and proliferation of cytotoxic CD8+ T cells argues that the mechanism of action of Kynase-PEG acts by relieving the immune suppression that normally occurs in the tumor microenvironment as a consequence of Kyn accumulation. Thus, KYNase-PEG represent a “first in class” immune checkpoint enzyme. Progress in developing a clinical candidate enzyme will be discussed. Citation Format: Everett Stone, Nicholas Marshall, Moses Donkor, Kendra Triplett, John Blazek, Todd Triplett, Lauren Ehrlich, George Georgiou. Depletion of kynurenine using an engineered therapeutic enzyme potently inhibits cancer immune checkpoints both as a monotherapy and in combination with anti-PD-1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-226. doi:10.1158/1538-7445.AM2015-LB-226

CD25 identifies a subset of CD4⁺FoxP3⁻ TIL that are exhausted yet prognostically favorable in human ovarian cancer.

CD25, the alpha subunit of the IL2 receptor, is a canonical marker of regulatory T cells (Treg) and hence has been implicated in immune suppression in cancer. However, CD25 is also required for optimal expansion and activity of effector T cells in peripheral tissues. Thus, we hypothesized that CD25, in addition to demarcating Tregs, might identify effector T cells in cancer. To investigate this possibility, we used multiparameter flow cytometry and IHC to analyze tumor-infiltrating lymphocytes (TIL) in primary high-grade serous carcinomas, the most common and fatal subtype of ovarian cancer. CD25 was expressed primarily by CD4⁺ TIL, with negligible expression by CD8⁺ TIL. In addition to conventional CD25⁺FoxP3⁺ Tregs, we identified a subset of CD25⁺FoxP3⁻ T cells that comprised up to 13% of CD4⁺ TIL. In tumors with CD8⁺ TIL, CD25⁺FoxP3⁻ T cells showed a strong positive association with patient survival (HR, 0.56; P = 0.02), which exceeded the negative effect of Tregs (HR, 1.55; P = 0.09). Among CD4⁺ TIL subsets, CD25⁺FoxP3⁻ cells expressed the highest levels of PD-1. Moreover, after in vitro stimulation, they failed to produce common T-helper cytokines (IFNγ, TNFα, IL2, IL4, IL10, or IL17A), suggesting that they were functionally exhausted. In contrast, the more abundant CD25⁻FoxP3⁻ subset of CD4⁺ TIL expressed low levels of PD-1 and produced T-helper 1 cytokines, yet conferred no prognostic benefit. Thus, CD25 identifies a subset of CD4⁺FoxP3⁻ TIL that, despite being exhausted at diagnosis, have a strong, positive association with patient survival and warrant consideration as effector T cells for immunotherapy.

The influence of total intravenous anaesthesia and isoflurane anaesthesia on plasma interleukin-6 and interleukin-10 concentrations after colorectal surgery for cancer

Although it has been demonstrated that both surgery and anaesthesia induce immune suppression, it remains unclear whether there are differences between anaesthetic techniques in inducing immune suppression in cancer patients. The aim of this present study was to compare the effects of total intravenous anaesthesia (TIVA) and isoflurane anaesthesia on plasma concentrations of interleukins IL-6 and IL-10 in patients undergoing surgery for colorectal cancer. A randomised, controlled, open-label study. University hospital. Seventy patients undergoing open colorectal surgery with tumour resection were randomised prospectively into one of two groups; 60 patients completed the study. Group 1 (n = 30) received TIVA and group 2 (n = 30) received isoflurane. Propofol infusion rate and inspired concentration of isoflurane were titrated to achieve bispectral index values of 40 to 55. Plasma concentrations of IL-6 and IL-10 were measured preoperatively, before surgical incision and at 2 and 24 h postoperatively. The area under the curve (AUC) for IL-6 and IL-10 over 24 h and plasma interleukin concentrations at each time point were compared between the groups. Median (range) AUC for IL-6 was 4657 (1219 to 8427) pg h ml in the TIVA group and 5349 (839 to 8126) pg h ml in the isoflurane group. For IL-10, AUC was 1165 (344 to 5258) pg h ml in the TIVA group and 1405 (463 to 8161) pg h ml in the isoflurane group. When comparing interleukin concentrations between study groups at different time intervals, there were no significant differences in plasma concentrations of IL-6 or IL-10. Intragroup comparisons revealed that IL-6 and IL-10 concentrations were significantly increased 2 and 24 h postoperatively in both groups when compared with their baseline values (P < 0.01 and P < 0.01 at 2 and 24 h for the TIVA group and isoflurane group, respectively). We found no significant differences between the effects of TIVA and isoflurane anaesthesia on plasma concentrations of IL-6 and IL-10 after colorectal cancer surgery during the first 24 h postoperatively. Further studies are needed to determine differences in long-term outcome of these patients. Clinicaltrials.gov identifier: NCT01902849. The study was registered retrospectively.

Mechanisms of TIGIT-driven immune suppression in cancer

Meeting abstracts TIGIT is a co-inhibitory molecule that limits T cell proliferation and activation. TIGIT expression has been recently shown to identify a subset of regulatory T cells (Treg) that specifically suppresses Th1 and Th17 responses; however its role in tumor immunity has not been

Silibinin inhibits accumulation of myeloid-derived suppressor cells and tumor growth of murine breast cancer.

Myeloid-derived suppressor cells (MDSC)s increase in blood and accumulate in the tumor microenvironment of tumor-bearing animals, contributing to immune suppression in cancer. Silibinin, a natural flavonoid from the seeds of milk thistle, has been developed as an anti-inflammatory agent and supportive care agent to reduce the toxicity of cancer chemotherapy. The goals of this study were to evaluate the effect of silibinin on MDSCs in tumor-bearing mice and antitumor activity of silibinin in a mouse model of breast cancer. 4T1 luciferase-transfected mammary carcinoma cells were injected into in the mammary fat pad female BALB/c mice, and female CB17-Prkdc Scid/J mice. Silibinin treatment started on day 4 or day 14 after tumor inoculation continued every other day. Tumor growth was monitored by bioluminescent imaging (BLI) measuring total photon flux. Flow cytometry measured total leukocytes, CD11b+ Gr-1+ MDSC, and T cells in the blood and tumors of tumor-bearing mice. The effects of silibinin on 4T1 cell viability in vitro were measured by BLI. Treatment with silibinin increased overall survival in mice harboring tumors derived from the 4T1-luciferase breast cancer cell line, and reduced tumor volumes and numbers of CD11b+Gr-1+ MDSCs in the blood and tumor, and increased the content of T cells in the tumor microenvironment. Silibinin failed to inhibit tumor growth in immunocompromised severe combined immunodeficiency mice, supporting the hypothesis that anticancer effect of silibinin is immune-mediated. The antitumor activity of silibinin requires an intact host immune system and is associated with decreased accumulation of blood and tumor-associated MDSCs.

Effects of Notch Signaling on Regulation of Myeloid Cell Differentiation in Cancer

Functionally altered myeloid cells play an important role in immune suppression in cancer, in angiogenesis, and in tumor cells' invasion and metastases. Here, we report that inhibition of Notch signaling in hematopoietic progenitor cells (HPC), myeloid-derived suppressor cells (MDSC), and dendritic cells is directly involved in abnormal myeloid cell differentiation in cancer. Inhibition of Notch signaling was caused by the disruption of the interaction between Notch receptor and transcriptional repressor CSL, which is normally required for efficient transcription of target genes. This disruption was the result of serine phosphorylation of Notch. We demonstrated that increased activity of casein kinase 2 (CK2) observed in HPC and in MDSC could be responsible for the phosphorylation of Notch and downregulation of Notch signaling. Inhibition of CK2 by siRNA or by pharmacological inhibitor restored Notch signaling in myeloid cells and substantially improved their differentiation, both in vitro and in vivo. This study demonstrates a novel mechanism regulation of Notch signaling in cancer. This may suggest a new perspective for pharmacological regulation of differentiation of myeloid cells in cancer.

Subpopulations of myeloid‐derived suppressor cells impair T cell responses through independent nitric oxide‐related pathways

The accumulation of myeloid-derived suppressor cells (MDSC) in tumor-bearing hosts is a hallmark of malignancy-associated inflammation and a major mediator for the induction of T cell suppression in cancer. MDSC can be divided phenotypically into granulocytic (G-MDSC) and monocytic (Mo-MDSC) subgroups. Several mechanisms mediate the induction of T cell anergy by MDSC; however, the specific role of these pathways in the inhibitory activity of MDSC subpopulations remains unclear. Therefore, we aimed to determine the effector mechanisms by which subsets of tumor-infiltrating MDSC block T cell function. We found that G-MDSC had a higher ability to impair proliferation and expression of effector molecules in activated T cells, as compared to Mo-MDSC. Interestingly, both MDSC subgroups inhibited T cells through nitric oxide (NO)-related pathways, but expressed different effector inhibitory mechanisms. Specifically, G-MDSC impaired T cells through the production of peroxynitrites (PNT), while Mo-MDSC suppressed by the release of NO. The production of PNT in G-MDSC depended on the expression of gp91(phox) and endothelial NO synthase (eNOS), while inducible NO synthase (iNOS) mediated the generation of NO in Mo-MDSC. Deletion of eNOS and gp91(phox) or scavenging of PNT blocked the suppressive function of G-MDSC and induced anti-tumoral effects, without altering Mo-MDSC inhibitory activity. Furthermore, NO-scavenging or iNOS knockdown prevented Mo-MDSC function, but did not affect PNT production or suppression by G-MDSC. These results suggest that MDSC subpopulations utilize independent effector mechanisms to regulate T cell function. Inhibition of these pathways is expected to specifically block MDSC subsets and overcome immune suppression in cancer.

Current status of interleukin-10 and regulatory T-cells in cancer

Tumor growth elicits antigen-specific cytotoxic as well as immune suppressive responses. Interleukin-10 (IL-10) is a key immune-suppressive cytokine produced by regulatory T-cells and by helper T-cells. Here, we review pleiotropic functions of IL-10 that impact the immune pathology of cancer. The role of IL-10 in cancer has become less certain with the knowledge of its immune stimulatory functions. IL-10 is needed for T-helper cell functions, T-cell immune surveillance, and suppression of cancer-associated inflammation. By promoting tumor-specific immune surveillance and hindering pathogenic inflammation, IL-10 is emerging as a key cytokine in the battle of the host against cancer. IL-10 functions at the cross-roads of immune stimulation and immune suppression in cancer. Immunological mechanisms of action of IL-10 can be ultimately exploited to develop novel and effective cancer therapies.

Immunity of human epithelial ovarian carcinoma: the paradigm of immune suppression in cancer

Epithelial ovarian cancer (EOC) is a significant cause of cancer-related mortality in women, and there has been no substantial decrease in the death rates due to EOC in the last three decades. Thus, basic knowledge regarding ovarian tumor cell biology is urgently needed to allow the development of innovative treatments for EOC. Traditionally, EOC has not been considered an immunogenic tumor, but there is evidence of an immune response to EOC in patients. Clinical data demonstrate that an antitumor immune response and immune evasion mechanisms are correlated with a better and lower survival, respectively, providing evidence for the immunoediting hypothesis in EOC. This review focuses on the immune response and immune suppression in EOC. The immunological roles of chemotherapy and surgery in EOC are also described. Finally, we detail pilot data supporting the efficiency of immunotherapy in the treatment of EOC and the emerging concept that immunomodulation aimed at counteracting the immunosuppressive microenvironment must be associated with immunotherapy strategies.

Abstract 4985: Expression of FasL on the tumor endothelium establishes a selective immune barrier in ovarian cancer.

Abstract There is an intimate relationship between angiogenesis and immune suppression in cancer, and anti-angiogenesis strategies can complement cancer immunotherapy. Previous research has implicated the tumor endothelium as a critical regulator of anti-tumor immunity, and our current work establishes a new role for the tumor endothelium as a critical barrier to T cell infiltration mediated through FasL. FasL is an important regulator of immunity and apoptosis, and we have found that, in contrast to the endothelium of normal tissues, the endothelium of multiple tumor types expressed FasL. FasL expression was restricted to tumor islets, and hypoxic tumor cell-conditioned media induced FasL on endothelial cells in vitro. Combination treatment with proangiogenic growth factors VEGF and PGE2 induced endothelial FasL expression in vitro, and COX1 and VEGF expression were associated with endothelial expression of FasL in ovarian cancer patients. Combinatorial blockade of VEGF and COX enzymes reduced endothelial FasL expression in mice, decreased tumor volume, and increased the CD8/FoxP3 ratio. In patients, low endothelial FasL expression was associated with a high CD8/Foxp3 ratio and increased survival. We found that endothelial FasL contributes to an unfavorable CD8/FoxP3 ratio through preferential killing of tumor-reactive CD8 T cells relative to Tregs. Increased intratumoral CD8/FoxP3 ratio was also observed in tumors grown on gld and lpr mice, but tumor growth was not robustly inhibited in these mice. Tumor control was dependent on the induction of an anti-tumor immune response, and CD8 depletion reversed the effects of VEGF and COX blockade. Additionally, combinatorial VEGF and COX blockade, tumors grown on gld mice, or the use of anti-FasL antibody all enhanced infiltration of adoptively transferred anti-tumor T cells and reduced apoptosis of transferred cells. Together, this work lends mechanistic insights into existing observations regarding T cells and tumor angiogenesis, and provides additional evidence for the paradigm that angiogenesis and immune suppression are intricately linked. Additionally, this work provides new strategies for cancer immunotherapy that may result in superior tumor control in patients. Citation Format: Greg Motz, George Coukos. Expression of FasL on the tumor endothelium establishes a selective immune barrier in ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4985. doi:10.1158/1538-7445.AM2013-4985 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Cod glycopeptide with picomolar affinity to galectin-3 suppresses T-cell apoptosis and prostate cancer metastasis

Cancer metastasis and immune suppression are critical issues in cancer therapy. Here, we show that a β-galactoside-binding lectin [galectin-3 (gal3)] that recognizes the Thomsen-Friedenreich disaccharide (TFD, Galβ1,3GalNAc) present on the surface of most cancer cells is involved in promoting angiogenesis, tumor-endothelial cell adhesion, and metastasis of prostate cancer cells, as well as evading immune surveillance through killing of activated T cells. To block gal3-mediated interactions, we purified a glycopeptide from cod (designated TFD100) that binds gal3 with picomolar affinity. TFD100 blocks gal3-mediated angiogenesis, tumor-endothelial cell interactions, and metastasis of prostate cancer cells in mice at nanomolar levels. Moreover, apoptosis of activated T cells induced by either recombinant gal3 or prostate cancer patient serum-associated gal3 was inhibited at nanomolar concentration of TFD100. Because the gal3-TFD interaction is a key factor driving metastasis in most epithelial cancers, this high-affinity TFD100 should be a promising antimetastatic agent for the treatment of various cancers, including prostate adenocarcinoma.

Molecular Pathways: Tumor-Infiltrating Myeloid Cells and Reactive Oxygen Species in Regulation of Tumor Microenvironment

Tumor-associated myeloid cells are the major type of inflammatory cells involved in the regulation of antitumor immune responses. One key characteristic of these cells is the generation of reactive oxygen (ROS) and reactive nitrogen species (RNS) in the tumor microenvironment. Recent studies have shown the important role of ROS and RNS, especially peroxynitrite, in immune suppression in cancer. ROS and RNS are involved in induction of antigen-specific T-cell tolerance, inhibition of T-cell migration to the tumor site, and tumor cell evasion of recognition by cytotoxic T cells. In preclinical settings, a number of potential therapeutic agents showed activity in blocking ROS/RNS in cancer and in improving the efficacy of cancer immune therapy. A better understanding of ROS/RNS-associated pathways in myeloid cells will help to identify more specific and direct targets to facilitate the development of more effective immune therapy for cancer.

PGE2-Driven Induction and Maintenance of Cancer-Associated Myeloid-Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSCs) are critical mediators of tumor-associated immune suppression, with their numbers and activity strongly increased in most human cancers and animal models. MDSCs suppress anti-tumor immunity through multiple mechanisms, including the manipulation of arginine and tryptophan metabolism by such factors as arginase (Arg), inducible nitric oxide synthase (iNOS/NOS2), and indoleamine-2,3-dioxygenase (IDO). Prostaglandin E2 (PGE2), a mediator of chronic inflammation and tumor progression, has emerged as a key molecule in MDSC biology. PGE2 promotes MDSC development and their induction by additional factors, directly suppresses T cell immune responses and participates in the induction of other MDSC-associated suppressive factors, including Arg, iNOS and IDO. It further promotes MDSC recruitment to tumor environments through the local induction of CXCL12/SDF-1 and the induction and stabilization of the CXCL12 receptor, CXCR4, on tumor-associated MDSCs. The establishment of a positive feedback loop between PGE2 and cyclooxygenase 2 (COX-2), the key regulator of PGE2 synthesis, stabilizes the MDSC phenotype and is required for their suppressive function. The central role of PGE2 in MDSC biology provides for a feasible target for counteracting MDSC-mediated immune suppression in cancer.

Exosome removal as a therapeutic adjuvant in cancer

Exosome secretion is a notable feature of malignancy owing to the roles of these nanoparticles in cancer growth, immune suppression, tumor angiogenesis and therapeutic resistance. Exosomes are 30–100 nm membrane vesicles released by many cells types during normal physiological processes. Tumors aberrantly secrete large quantities of exosomes that transport oncoproteins and immune suppressive molecules to support tumor growth and metastasis. The role of exosomes in intercellular signaling is exemplified by human epidermal growth factor receptor type 2 (HER2) over-expressing breast cancer, where exosomes with the HER2 oncoprotein stimulate tumor growth and interfere with the activity of the therapeutic antibody Herceptin®. Since numerous observations from experimental model systems point toward an important clinical impact of exosomes in cancer, several pharmacological strategies have been proposed for targeting their malignant activities. We also propose a novel device strategy involving extracorporeal hemofiltration of exosomes from the entire circulatory system using an affinity plasmapheresis platform known as the Aethlon ADAPT™ (adaptive dialysis-like affinity platform technology) system, which would overcome the risks of toxicity and drug interactions posed by pharmacological approaches. This technology allows affinity agents, including exosome-binding lectins and antibodies, to be immobilized in the outer-capillary space of plasma filtration membranes that integrate into existing kidney dialysis systems. Device therapies that evolve from this platform allow rapid extracorporeal capture and selective retention of target particles < 200 nm from the entire circulatory system. This strategy is supported by clinical experience in hepatitis C virus-infected patients using an ADAPT™ device, the Hemopurifier®, to reduce the systemic load of virions having similar sizes and glycosylated surfaces as cancer exosomes. This review discusses the possible therapeutic approaches for targeting immune suppressive exosomes in cancer patients, and the anticipated significance of these strategies for reversing immune dysfunction and improving responses to standard of care treatments.

Immunoactivative role of indoleamine 2,3‑dioxygenase in gastric cancer cells in vitro

To study the role of indoleamine 2,3-dioxygenase (IDO) in immune response and immune escape in gastric cancer, the human IDO gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR) and inserted into the pIRES2-EGFP vector to construct the IDO expression vector (pIRES2-EGFP-IDO). BGC-823 cells were transfected with the vector by electroporation and selected stable expression with G418. IDO expression was determined by RT-PCR and Western blot analysis. The enzymatic activity of IDO was estimated by determining tryptophan and kynurenine concentrations in the cell culture medium by an amino acid analyzer. To assess the effect of IDO on T cell-mediated cytotoxicity and proliferation, T cells from patients with gastric cancer were co-cultured with the IDO-transfected BGC-823 cells in the presence or absence of 1-MT, a competitive inhibitor of IDO. Cells transfected with the vector expressed high levels of IDO mRNA and protein, and a significantly higher level of kynurenic acid was detected in the culture medium of the transfected cells compared to the non-transfected cells (P<0.001). T cells co-cultured with the IDO-transfected cells exhibited significantly lower cytotoxicity compared to the control group (P<0.05). Additionally, IDO-transfected cells treated with 1-MT exhibited higher toxicity compared to the untreated IDO-transfected cells (P<0.01). We conclude that IDO plays a key role in gastric cancer immune suppression, possibly by inhibiting T cell-mediated cytotoxicity and proliferation in vitro.

Functional Screening Studies Identify Combinational Activity of PD-L1 and CD200 In Mediating Dysfunctional T Cell Immunological Synapse Formation In Chronic Lymphocytic Leukemia

AbstractAbstract 696The ability of cancer cells to modulate the immune microenvironment is now recognized as an important hallmark of disease pathophysiology. Identifying the molecular mechanisms of cancer immune suppression in the laboratory is key to the design of more effective immunotherapeutic treatment strategies. We previously demonstrated that chronic lymphocytic leukemia (CLL) cells induce alterations in global gene expression profiles in patient CD4 and CD8 T cells, and a profound T cell immunological synapse formation defect that can be reversed with lenalidomide (J Clin Invest. 2005;115(7):1797-1805, and 2008;118(7):2427-2437). Here we used small interfering RNA (siRNA) with a 2-part functional screen to identify key CLL cell molecules inducing T cell immune suppression. siRNA treated tumor cells were cocultured in direct contact with healthy allogeneic T cells for 24 hours, T cells purified from coculture and used in cell conjugation immune synapse assays with superantigen-pulsed third party B cells as antigen-presenting cells (APCs). Confocal microscopy and image analysis software was used to quantify the mean area of T cell F-actin immune synapse formation events from each experimental cell population. Treatment of the CLL cell line MEC-1 with either TNFα, TGFβ, IL-10, or IL-6 siRNA identified no gain in subsequent CD3 T cell immune synapse function compared to control non-targeting siRNA or untreated CLL cells. However, CD200 or programmed death 1 (PD1) ligand 1 (PD-L1, CD274) siRNA treatment significantly enhanced (P < .01) subsequent T cell synapse formation events with APCs (comparable to positive control experiments blocking tumor cell:T cell direct contact with ICAM-1 siRNA, or primary coculture of T cells with allogeneic healthy donor B cells). Primary CLL patient cells (n=10) were treated with individual or pooled neutralizing antibodies, or siRNA, targeting PD-L1, CD200, or cytokines. This analysis revealed that counteracting the combined activity of PD-L1, CD200 and TGFβ exhibited the most pronounced repair of subsequent T cell synapse function compared to control treated tumor cells (P < .01). These data suggest that CLL-released cytokines such as TGFβ contribute to, but are not essential for the T cell synapse defect. We also identified that blocking the T cell receptors PD-1, CD200-R and TGFβ-R1 with neutralizing antibodies prevents CLL inhibitory signaling (P < .01) compared to isotype control IgG treated T cells in contact with tumor cells. We further show that knock-down of PD-L1, CD200 and TGFβ on ex vivo CLL cells prevents inhibitory CD4 and CD8 T cell synapse function compared to control siRNA (P < .01) using the Eμ-TCL1 mouse model of CLL. The addition of lenalidomide (1μM) in ex vivo CLL cell:T cell coculture assays significantly increased (P < .01) subsequent T cell synapse function compared to untreated vehicle control experiments. Flow cytometric analysis identified that lenalidomide down-regulates both CLL expressed PD-L1 and CD200 ligands, and T cell cognate receptor PD1 and CD200R expression during intercellular contact interactions. Moreover, subsequent effector T cell killing function was significantly enhanced (P < .05) following antibody blockade of CLL cell PD-L1 and CD200 with or without lenalidomide treatment during primary coculture with CD8 T cells. We are currently investigating the expression and activity of PD-L1, CD200, and other co-inhibitory molecules in CLL and other haematological and solid malignancies, using patient tissue microarray analysis and confocal co-localization analysis. This work is identifying common inhibitory ligands utilized by tumor cells to suppress T cell synapse function. These results provide important mechanistic insight into immune suppression in CLL and the action of lenalidomide, and identify co-inhibitory ligands as potential immunotherapeutic targets to repair T cell function. Disclosures:Gribben:Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.

L-Arginine Deprivation Regulates Cyclin D3 mRNA Stability in Human T Cells by Controlling HuR Expression

Myeloid-derived suppressor cells are a major mechanism of tumor-induced immune suppression in cancer. Arginase I-producing myeloid-derived suppressor cells deplete l-arginine (L-Arg) from the microenvironment, which arrests T cells in the G(0)-G(1) phase of the cell cycle. This cell cycle arrest correlated with an inability to increase cyclin D3 expression resulting from a decreased mRNA stability and an impaired translation. We sought to determine the mechanisms leading to a decreased cyclin D3 mRNA stability in activated T cells cultured in medium deprived of L-Arg. Results show that cyclin D3 mRNA instability induced by L-Arg deprivation is dependent on response elements found in its 3'-untranslated region (UTR). RNA-binding protein HuR was found to be increased in T cells cultured in medium with L-Arg and bound to the 3'-untranslated region of cyclin D3 mRNA in vitro and endogenously in activated T cells. Silencing of HuR expression significantly impaired cyclin D3 mRNA stability. L-Arg deprivation inhibited the expression of HuR through a global arrest in de novo protein synthesis, but it did not affect its mRNA expression. This alteration is dependent on the expression of the amino acid starvation sensor general control nonderepressible 2 kinase. These data contribute to an understanding of a central mechanism by which diseases characterized by increased arginase I production may cause T cell dysfunction.

Myeloid-Derived Suppressor Cells in Human Cancer

Myeloid-derived suppressor cells are one of the major factors responsible for immune suppression in cancer. They also contribute to limited efficacy of current vaccination strategies. Here, we give an overview of the myeloid-derived suppressor cells field focusing primarily on the studies in cancer patients and current and future therapeutic options targeting these cells.