Tumor-induced Immune Suppression Research Articles

Abstract 461: Inhibition of HMGB1 delays tumor progression, reduces MDSC-mediated immune suppression, and diminishes MDSC-macrophage cross-talk interaction.

Abstract Cancer immunotherapy aims to harness the power of the immune system to fight advanced stages of cancer. A main hindrance to cancer immunotherapy is tumor-induced immune suppression. Within the tumor microenvironment are a plethora of immune suppressive cells including Myeloid-Derived Suppressor Cells (MDSC). MDSC are present in nearly all types of cancer blocking T cell activation and shifting immunity towards a type 2 tumor-promoting phenotype through cross-talk with macrophages that enhances the secretion of pro-tumor molecules such as IL-10, and simultaneously drops IL-12 production by macrophages. MDSC accumulation is induced by pro-inflammatory cytokines including IL-1β, IL-6 and VEGF as well as alarmins such as S100A8/9. Because the alarmin HMGB1 is increased in many cancers, we are determining if HMGB1 drives MDSC. Inhibition of HMGB1 in vitro by chemical inhibitors Glycyrrhizin and Ethyl Pyruvate decreased cross-talk-induced IL-10 production by MDSC and IL-6 production by macrophages. Ethyl Pyruvate also reduced MDSC suppressive capacity in transgenic T cell suppression assays, suggesting that HMGB1 regulates multiple aspects of MDSC function. Treatment of BALB/c mice with 4T1 mammary carcinoma with HMGB1 inhibitors Glycyrrhizin and Ethyl Pyruvate reduced lung metastases and did not impact primary tumor progression. Treatment of C57BL/6 mice with MC38 colon carcinoma with an HMGB1 neutralizing antibody (2G7) reduced MDSC levels in the blood, spleen, and tumor. HMGB1 consists of two domains, the pro-inflammatory B box domain and an anti-inflammatory A box domain. Treatment of C57BL/6 mice with MC38 tumor with A box significantly reduced primary tumor growth and decreased MDSC levels in the blood. These findings indicate that full-length HMGB1 drives inflammation and tumor progression, while the anti-inflammatory A box domain counteracts full-length HMGB1 and reduces tumor growth and MDSC. These results suggest that HMGB1 drives MDSC accumulation and tumor progression and that A box can be used to target MDSC-mediated tumor-induced immune suppression. (Research supported by NIH RO1CA115880 and RO1CA84232) Citation Format: Katherine Parker, Suzanne Rosenberg, Pratima Sinha, Huan Yang, Kevin Tracey, Jianhua Li. Inhibition of HMGB1 delays tumor progression, reduces MDSC-mediated immune suppression, and diminishes MDSC-macrophage cross-talk interaction. [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 461. doi:10.1158/1538-7445.AM2013-461

Abstract 1249: Potent immunomodulatory effects of lenalidomide enhance the effect of a therapeutic vaccine against established lymphoma in mice .

Abstract Lenalidomide is an effective therapeutic agent with direct inhibitory effects on malignant B- and plasma cells and immunomodulatory effects on the T cell activation. The dual function of lenalidomide makes it an appealing candidate for combination with other novel cancer agents. We investigated the immune stimulatory effects of lenalidomide, administrated to mice in doses which provided comparable pharmacokinetics to human patients, on the potency of a novel fusion DNA lymphoma vaccine. The combination protected mice from lethal challenge with syngeneic A20 murine lymphoma, resulting in significantly improved survival compared with vaccine or lenalidomide alone and induced immune memory. In vivo depletion experiments demonstrated a requirement for effector CD8+ and CD4+ T cells. Surprisingly, lenalidomide alone was associated with reduced numbers of systemic immune suppressive cells (MDSC/Treg) in tumor-bearing, but not naïve mice, an effect that was independent of simple tumor burden reduction. Finally, the combination of lenalidomide and vaccine produced significantly improved survival in mice with 7 day established tumors. These results demonstrated the dual effect of lenalidomide on enhancing antigen-specific T-cell immunity and a novel mechanism of action reversing tumor-induced immune suppression, which makes it ideal for combination with vaccines as an immune adjuvant. Citation Format: Ippei Sakamaki, Larry W. Kwak, Soung-chul Cha, Qing Yi, Beatrisa Lerman, Jian Chen, Sekhar Surapaneni, Scott Bateman, Hong Qin. Potent immunomodulatory effects of lenalidomide enhance the effect of a therapeutic vaccine against established lymphoma in mice . [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 1249. doi:10.1158/1538-7445.AM2013-1249

Abstract 492: Ibrutinib inhibits Interleukin-2 inducible kinase, driving a Th1 selective pressure in human leukemia patients that serves to alleviate tumor-induced immunosupression.

Abstract Interleukin-2 Inducible Kinase (ITK) is an appealing therapeutic target given its critical role in polarizing T-cell immunity against various infectious, autoimmune, and neoplastic diseases. ITK activity promotes Th2 immunity, thereby subverting healthy Th1-based immune surveillance. Th2 CD4 T-cells depend on ITK for activation, whereas Th1 and CD8 T-cells have compensatory resting lymphocyte kinase (RLK) that conducts T-cell receptor activation in the absence of ITK. Similar to many other tumors, chronic lymphocytic leukemia (CLL) directs an aberrant Th2 bias that drives immune evasion, promotes a supportive microenvironment, and cripples adaptive immunity. In CLL, this immune suppression leads to severe infections which are a leading cause of death. Although ITK inhibitors have the promise of alleviating tumor-induced immune suppression, to date no clinically viable ITK inhibitor exists. Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) with outstanding activity and tolerability in B-cell malignancies. The striking homology between BTK and ITK combined with our in silico docking studies and in vitro kinase inhibition profiles led us to identify ibrutinib as the first clinically viable irreversible ITK inhibitor. We further confirmed both molecular and functional outcomes using Jurkat T-cells and in vitro polarized Th1 and Th2 CD4 T-cells. We found that mutation of the ITK-Cys442 covalent binding residue for ibrutinib alleviated molecular inhibition. We also demonstrated that Th1 and CD8 T-cell restricted expression of RLK provides a compensatory platform for T-cell activation, offering a molecular explanation for the selective outgrowth of cytotoxic Th1-biased immunity. This effect was confirmed using T-cells directly derived from CLL patients. To clearly demonstrate this effect in vivo, we utilized the Leishmania major model of Th2-mediated infection. Ibrutinib enhanced resistance to the parasite by eliciting robust Th1 immunity. To demonstrate a direct clinical relevance in the setting of CLL we utilized a novel listeriosis/leukemia mouse model. In this model, we observed complete recovery of functional immunity, with all ibrutinib-treated mice surviving a potentially lethal Listeria monocytogenes infection. We further confirmed irreversible binding of ITK in patients currently receiving ibrutinib as part of a clinical trial. Finally, we found that ibrutinib inhibited phosphorlyation of ITK's direct downstream target PLCγ1 in CD4 T-cells. Together, our results provide novel molecular insights into the mechanism of action of ibrutinib in the context of Th2-biased immunosuppressive leukemia. We also postulate that ibrutinib's irreversible ITK inhibitory effects may prove beneficial in a number of other autoimmune, inflammatory, parasitic, and viral diseases. Citation Format: Jason A. Dubovsky, Kyle A. Beckwith, Gayathri Natarajan, Jennifer A. Woyach, Samantha Jaglowski, Joshua Hessler, Ta-Ming Liu, Betty Y. Chang, Karilyn M. Larkin, Matthew R. Stefanovski, Frank W. Frissora, Lisa L. Smith, Kelly A. Smucker, Joseph M. Flynn, Jeffrey A. Jones, Leslie A. Andritsos, Kami Maddocks, Amy M. Lehman, Richard Furman, Jeff Sharman, Anjali Mishra, Michael A. Caligiuri, Abhay R. Satoskar, Joseph J. Buggy, Natarajan Muthusamy, Amy J. Johnson, John C. Byrd. Ibrutinib inhibits Interleukin-2 inducible kinase, driving a Th1 selective pressure in human leukemia patients that serves to alleviate tumor-induced immunosupression. [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 492. doi:10.1158/1538-7445.AM2013-492

P23: Global S-nitrosylation analysis provides insight into tumor-induced immune suppression

To explore the functions of nitric oxide (NO) in immune regulation, we performed a large-scale, in vivo, site-specific analysis of S-nitrosylated proteins, from the spleens of naive (NTB) and mammary tumor bearing (TB) mice, yielding a dataset of 3352 S-nitrosylated sites on 1690 proteins. In TB mice S-nitrosylation mainly affects DNA replication/recombination/repair, nitrogen compound metabolic processes, translation, organelle organization and protein kinase cascades; whereas proteolysis, cell adhesion, protein transport and protein folding are primarily targeted by S-nitrosylation in naive mice. The GM-CSF pathway which controls normal hematopoiesis and myeloid differentiation is targeted by S-nitrosylation in TB mice. S-nitrosylation of DNA polymerase delta inhibitory to its function is also observed. Our data demonstrate the broad immune regulatory role of NO in both physiological and pathophysiological processes. Disclosure Nothing to disclose.

Targeting the EP1 receptor reduces Fas ligand expression and increases the antitumor immune response in an in vivo model of colon cancer

Despite studies demonstrating that inhibition of cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2 ) has significant chemotherapeutic benefits in vitro and in vivo, inhibition of COX enzymes is associated with serious gastrointestinal and cardiovascular side effects, limiting the clinical utility of these drugs. PGE2 signals through four different receptors (EP1-EP4) and targeting individual receptor(s) may avoid these side effects, while retaining significant anticancer benefits. Here, we show that targeted inhibition of the EP1 receptor in the tumor cells and the tumor microenvironment resulted in the significant inhibition of tumor growth in vivo. Both dietary administration and direct injection of the EP1 receptor-specific antagonist, ONO-8713, effectively reduced the growth of established CT26 tumors in BALB/c mice, with suppression of the EP1 receptor in the tumor cells alone less effective in reducing tumor growth. This antitumor effect was associated with reduced Fas ligand expression and attenuated tumor-induced immune suppression. In particular, tumor infiltration by CD4(+) CD25(+) Foxp3(+) regulatory T cells was decreased, whereas the cytotoxic activity of isolated splenocytes against CT26 cells was increased. F4/80(+) macrophage infiltration was also decreased; however, there was no change in macrophage phenotype. These findings suggest that the EP1 receptor represents a potential target for the treatment of colon cancer.

Immunoediting and Antigen Loss: Overcoming the Achilles Heel of Immunotherapy with Antigen Non-Specific Therapies

Cancer immunotherapy has emerged as a mainstream therapy option in the battle against cancer. Pre-clinical data demonstrates the ability of immunotherapy to harness the immune system to fight disseminated malignancy. Clinical translation has failed to recapitulate the promising results of pre-clinical studies although there have been some successes. In this review we explore some of the short-comings of cancer immunotherapy that have limited successful clinical translation. We will give special consideration to what we consider the most formidable hurdle to successful cancer immunotherapy: tumor-induced immune suppression and immune escape. We will discuss the need for antigen-specific immune responses for successful immunotherapy but also consider the need for antigen specificity as an Achilles heel of immunotherapy given tumor heterogeneity, immune editing, and antigen loss. Finally, we will discuss how combinatorial strategies may overcome some of the pitfalls of antigen specificity and highlight recent studies from our lab which suggest that the induction of antigen non-specific immune responses may also produce robust anti-tumor effects and bypass the need for antigen specificity.

Dendritic Cell Plasticity in Tumor-Conditioned Skin: CD14+ Cells at the Cross-Roads of Immune Activation and Suppression

Tumors abuse myeloid plasticity to re-direct dendritic cell (DC) differentiation from T cell stimulatory subsets to immune-suppressive subsets that can interfere with anti-tumor immunity. Lined by a dense network of easily accessible DC the skin is a preferred site for the delivery of DC-targeted vaccines. Various groups have recently been focusing on functional aspects of DC subsets in the skin and how these may be affected by tumor-derived suppressive factors. IL-6, Prostaglandin-E2, and IL-10 were identified as factors in cultures of primary human tumors responsible for the inhibited development and activation of skin DC as well as monocyte-derived DC. IL-10 was found to be uniquely able to convert fully developed DC to immature macrophage-like cells with functional M2 characteristics in a physiologically highly relevant skin explant model in which the phenotypic and functional traits of “crawl-out” DC were studied. Mostly from mouse studies, the JAK2/STAT3 signaling pathway has emerged as a “master switch” of tumor-induced immune suppression. Our lab has additionally identified p38-MAPK as an important signaling element in human DC suppression, and recently validated it as such in ex vivo cultures of single-cell suspensions from melanoma metastases. Through the identification of molecular mechanisms and signaling events that drive myeloid immune suppression in human tumors, more effective DC-targeted cancer vaccines may be designed.

Abstract B17: Immunomodulation by tasquinimod: Combination with immunotherapy results in enhanced CD8 T cell responses and improved antitumor effects.

Abstract Purpose: The novel anti-tumor agent tasquinimod (ABR-215050) is being developed for treatment of metastatic castration-resistant prostate cancer. Proof of concept has been demonstrated in a Phase II trial by delaying disease progression and increasing overall survival, and enrollment of patients to a global pivotal Phase III trial (10TASQ10) is ongoing. Tasquinimod targets the tumor microenvironment resulting in anti-angiogenesis, inhibition of metastasis and immunomodulation. A target molecule of tasquinimod is the S100A9 protein which has been demonstrated to affect the accumulation and function of CD11b+Gr-1+ regulatory myeloid cell subsets. Given the major contribution of these cells to the immunosuppressive tumor environment, modulation of this cell population could contribute to the anti-tumor effects by tasquinimod and enhance the effects of cancer immunotherapy. In this study, we evaluated the immunomodulatory effects of tasquinimod when combined with tumor-targeted superantigens (TTS); an immunotherapeutic strategy which is highly dependent on activation of effector T cells directed against the tumor. Experimental design: The effect of tasquinimod treatment on CD11b+ myeloid cell populations was analyzed by flow cytometry in cell suspensions from spleens and from B16 tumors growing subcutaneously. In addition, B16-5T4 expressing tumors were treated with tasquinimod (30 mg/kg), the TTS fusion protein 5T4Fab-SEA (25 μg/kg), or the combination. Tumor growth and tumor-directed T cell responses were monitored. Results: The number of CD11b+ myeloid cells significantly increased in tumor bearing mice. Tasquinimod treatment inhibited tumor growth and reduced the CD11b+Ly6G-Ly6Chigh monocytic subpopulation of CD11b+Gr-1+ cells. In addition, less CD206-expressing tumor-associated macrophages were detected. Combining tasquinimod and TTS immunotherapy significantly enhanced the anti-tumor effects compared to the mono therapies. This was accompanied with increased numbers of TTS-activated CD8+ T cells in the tumors over a prolonged period of time. Conclusions: This study demonstrates that tasquinimod modulates the tumor infiltrating myeloid cell subsets and suppresses tumor growth. In line with an effect on the immunosuppressive tumor microenvironment, combining tasquinimod with the immunotherapy TTS gave rise to significantly improved anti-tumor effects. These data suggest that tasquinimod reduces tumor-induced immune suppression and that tasquinimod combined with immunotherapy could represent a novel and promising anti-cancer strategy. Citation Format: Anette Sundstedt, Anders Olsson, Martin Stenström, Mona Celander, Marie Törngren, Helena Eriksson, Gunnar Hedlund, David Liberg, Tomas Leanderson. Immunomodulation by tasquinimod: Combination with immunotherapy results in enhanced CD8 T cell responses and improved antitumor effects. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B17.

Combining DC-mediated immunotherapy with oncolytic virotherapy in the treatment of HGG

Oncolytic viruses (OV) are either naturally selective for neoplastic cells or are engineered to be so. Whereas conservative therapies use the unspecific genome vulnerability of highly proliferating cells to target malignancies, OV have the advantage of often being extremely specific in infecting tumor cells. This (intrinsic) selectivity combined with the demonstrated safety and lack of host toxicity in phase I clinical studies make OV very appealing as novel cancer therapeutics. Recently, Alkassar et al. have shown in vitro and in vivo that naturally occurring Reovirus, Parvovirus and Newcastle disease virus (NDV) strains mediate strong anti-tumoral effects on glioblastoma. We hypothesize that upon combining a systemic immunotherapy approach with preceding oncolytic virotherapy (OVT), as a means of lowering the tumor viability and volume and altering the local micro environment within the tumor, a better immune control can be induced. In the immunotherapy field, it is known that the immunosuppressive microenvironment induced by the tumor can profoundly suppress the antitumor immunity and therefore inhibit vaccine efficacy. Viral presence within the tumor does the opposite; acting as a danger signal to alert and activate the immune system, it can reduce or eliminate the tumor induced immune suppression. Furthermore, the direct tumor cell killing effects of OV can lower tumor burden prior to immunotherapy. Both active specific immunotherapy and OVT have shown promising results in preclinical and clinical studies but the possible additive or synergistic effects of these treatment strategies have not been studied. We aim to combine OVT with dendritic cell (DC)-mediated immunotherapy in a preclinical murine glioma model to demonstrate additive or synergistic effects. Our initial results show good susceptibility of the murine GL261 glioma cell line to cytotoxic killing by oncolytic Reovirus, Parvovirus and NDV in vitro. The strains were also combined to exploit the synergism recently demonstrated by Alkassar et al. Results indicate increased cytotoxic effects when Reovirus was combined with NDV or when Reovirus was combined with Parvovirus. GL261 cell infection capacity of the viruses was quantified by immunofluorescence imaging. In vivo, intracranial injection of Reovirus, Parvovirus, NDV or combinations of two viruses in healthy mice led to signs of inflammation in the brain, as visualized by MR imaging. Infiltration of inflammatory cells will be confirmed by histology. In tumor-bearing mice, prolonged survival and induction of long term survival was seen when mice were treated intra tumorally with Reovirus, Parvovirus or a combination of both. Similar results were found when tumor-bearing mice were treated with NDV and/or Reovirus. Toxicity of the viruses will be analyzed by histological analysis of brain and other vital organs.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 4405: Immune modulation and Treg suppression with metronomic cyclophosphamide enhances the anti-tumor effect of a therapeutic peptide vaccine in a murine model

Abstract In order to be optimally effective therapeutic cancer vaccines must be combined with immune modulatory approaches, particularly in advanced cancer where several different pathways of cancer induced immune suppression may be active. We have developed a potent cancer vaccine enhancement formulation named DepoVaxTM (DPX) that is particularly well suited for the delivery of peptide antigens. DPX is a novel vaccine platform comprised of liposomes in oil that is formulated with a TLR adjuvant, universal T-helper peptide and specific tumor peptide antigens. We have shown that DPX generates better immune responses than Montanide based emulsion formulations. A major obstacle to any cancer vaccine, however, is tumor induced immune suppression. Regulatory T cell (Treg) mediated immune suppression is an important mechanism of tumor immune evasion in many types of cancer including ovarian cancer. We sought to determine if combining a DPX-based peptide vaccine approach with immune modulation in the form of Treg suppression could enhance the anti-tumor effect of the vaccine. Metronomic cyclophosphamide (mCPA) treatment has been reported to selectively sustain reduced regulatory T cell numbers and activity. Using a murine tumor model (C3) we tested the benefit of combining mCPA and DPX vaccination for controlling well established subcutaneous solid tumors in vivo. We found that neither mCPA or DPX treatments alone were sufficient to eradicate advanced C3 tumors. However, combining mCPA with DPX vaccination provided effective tumor control. We further characterized the immune response by isolating and phenotyping tumor infiltrating lymphocytes and testing their immunogenicity by IFN-γ ELISPOT assay. Our results indicate that the DPX vaccine can increase the levels of antigen-specific CD8+ T cells and mCPA treatment reduces the CD4+FoxP3+ T regulatory cells. We conclude that immune responses generated by the DPX vaccine were more effective at controlling advanced tumors together with reduced Treg-mediated immune suppression. We have designed and initiated a phase I/II clinical trial to test a DPX vaccine targeting Survivin (DPX-Survivac), in combination with mCPA in ovarian cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4405. doi:1538-7445.AM2012-4405

Abstract 3537: Manipulation of local and systemic immune suppression by GPI-anchored immune stimulatory proteins

Abstract Each day in the United States alone, approximately 3400 people are diagnosed while another 1500 people die from cancer. Breast cancer is the most common form of cancer among females and accounts for the second leading cause of death among female cancer patients. In order to be effective in a therapeutic setting, current immunotherapies must be able to overcome the multiple layers of tumor-induced immune suppression such as modulation of MHC expression and costimulatory molecules (CD80/CD86), expression of inhibitory molecules PDL-1 and GITRL and the secretion of immunosuppressive factors such as VEGF and TGF-β into the tumor microenvironment. These immunosuppressive factors promote the recruitment, differentiation and expansion of regulatory immune cells such as myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs) into the tumor microenvironment. Using the 4T07 murine breast cancer model we investigated the effects of expressing GPI-anchored immune stimulatory molecules (GPI-ISMs), namely cytokines (IL-2, IL-12) and the costimulatory protein B7-1, on the surface of the tumor cells. BALB/c mice were challenged subcutaneously (s.c.) with either wild-type 4T07 cells (4T07-WT) or 4T07 cells expressing GPI-ISMs. We observed significant splenomegaly in the mice challenged with 4T07-WT cells relative to the mice challenged with 4T07 cells expressing GPI-ISMs. This observed splenomegaly correlated with tumor size and a 4-5 fold increase in the percentage of splenic CD11b+Gr1+ MDSCs indicating the role of active immune suppression in the tumorigenicity of 4T07 breast cancer cells. We then conducted studies to analyze the effect of GPI-ISMs on infiltrating cells into the tumor microenvironment as well as in the spleen and draining lymph nodes (dLNs). Three groups of mice were challenged (s.c.) with the following cells mixed in a 1:1 ratio with BD Matrigel™ (a solubilized basement membrane preparation derived from a mouse sarcoma): 4T07-WT, 4T07-B7/IL-12 or PBS (control). Seven days post challenge, the Matrigel/tumor, spleen and dLNs were harvested from the mice, digested and analyzed for cellular infiltrates by flow cytometry. We observed that the expression of GPI-ISMs on the surface of tumor cells led to reduced angiogenesis as evidenced by a reduced level of blood vessels and decreased presence of CD4+CD25+FOXP3+ regulatory T cells and CD11b+Gr1+ MDSCs locally at the tumor site and dLNs as well as systemically in the spleen. Additionally, there was a decrease in CD8+PD1+ exhausted T cells at the tumor site. Along with the inhibition of immune suppressive cell populations, the GPI-ISMs increased the presence of CD4+ and CD8+ T cells as well as dendritic cells and B cells. These observations suggest that components of the active immune suppression evident in this model can be inhibited by expressing GPI-ISMs on the surface of the 4T07 tumor cells and could be effective in a therapeutic setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3537. doi:1538-7445.AM2012-3537

Abstract 5417: Expansion of effector regulatory T-cells represents a novel and independent prognostic factor marking escape from immune surveillance in Myleodysplastic Syndrome

Abstract Myelodysplastic syndromes (MDS) refer to a group of pathophysiologically diverse hematopoietic neoplasms associated with cytopenias, myeloid dysplasia, and variable acute myeloid leukemia risk. Response to immunosuppressive therapies (IST) in a subset of younger patients classified as lower-risk by the International Prognostic Scoring System (IPSS) suggests that inflammation and immune reactivity in the bone marrow likely plays a role in early MDS pathogenesis. Conversely, higher-risk patients with more established disease do not respond to IST. Previous reports of increased regulatory T-cells (Tregs) in these patients suggest that immune escape may be required for progression to higher-risk disease. The distinct changes in the role of the immune system between lower and higher-risk disease suggests that MDS may progress according to established principles of cancer immuno-editing involving elimination, equilibrium and escape phases. Because Treg suppressive activity is dependant upon auto-antigen presentation, we hypothesized that analyzing the activation state of Tregs may better reflect the onset of tumor-induced immune suppression in MDS and have better prognostic utility than measuring total Treg numbers alone. Here, we use a panel of surface markers on Tregs that are normally employed to distinguish effector and memory populations among conventional T-cells and analyzed the resulting subpopulations for associations with clinical features including overall survival (OS) among patients predominantly classified as lower-risk. Abnormal numbers of Treg subtypes were seen in 18 (34.6%) of 52 patients compared to age-matched controls. The most prominent and unique change in MDS patients was an expansion of effector Tregs (TregEff) which was significantly associated with anemia (p=0.046) and reduced hemoglobin (p=0.038). This group of patients had increased blast percentage (p=0.006) and displayed worse OS by Cox-regression (HR 4.3, 95% CI 1.6-11.6, p=0.004) independent of IPSS (p=0.002) and the MD Anderson Scoring System (MDAS) (p=0.047). Increased TregEff numbers were not dependant on total Treg numbers. Rather, they came at the expense of central memory Tregs suggesting that Treg “class switching” may reflect active presentation of auto-antigens. When isolated, TregEff cells displayed increased suppressive capacity compared to other Treg subpopulations and heightened TregEff numbers were limited to patients with elevated blasts (tumor burden), suggesting a mechanistic link between immune-evasion and MDS progression. The MDAS significantly refined OS estimates (p<0.001) compared to IPSS. Using the MDAS, OS in higher-risk patients could be further stratified using TregEff numbers (p=0.018), suggesting that enumeration of TregEff cells may improve prognostication and impact therapeutic selection in MDS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5417. doi:1538-7445.AM2012-5417

Abstract 5405: Immuno-phenotyping reveals cell types present in circulation of breast cancer patients and elucidates their funstional potential

Abstract Myeloid derived suppressor cells (MDSC) have an identified role in suppressing anti-tumor immunity through Jak/Stat pathway activation and the generation of reactive oxygen species and arginase -1, thus promoting cancer progression. There is robust data on the identification of functionally suppressive MDSCs in preclinical models. However, such knowledge is underdeveloped in human cancers. Specifically, the clear identification of the true MDSC and their role in tumor immunoevasion, cancer progression and metastasis is undefined. Therefore, comprehensive identification of MDSCs and their ability to augment tumor-induced immune suppression in newly diagnosed early stage breast cancer is warranted to define their potential as a novel immunotherapeutic target. To delineate the role of MDSCs in newly diagnosed, untreated breast cancer and their tumor immunosuppressive effects in comparison to normal hosts, we conducted a prospective trial of stage I-IV breast cancer patients enrolled prior to initiation of systemic therapy and their age-matched normal donors. The following immune cell subsets were analyzed: regulatory T cells (CD3+/CD4+/CD25+/Foxp3+), MDSCs (Linlo/HLA-DR-/CD33+/CD11b+), and DCs (Lin-/CD14-CD4+/CD303+/CD1c+/CD141+). Functional assays of T cell activation and MDSCs mediated-T cell suppression were performed. We identified no significant differences in the percentage of regulatory T cells or mDC2 between breast cancer subjects and normal controls. In contrast, the percent of MDSC, pDCs and conventional DCs detected were suprisingly significantly higher in the healthy donor population as compared to breast cancer subjects. Breast cancer T cells showed reduced capability to respond to external stimulus as measured by up-regulation of CD25 and CD69 as well as T cell proliferation, although not statistically significant relative to normal controls. Breast cancer MDSC were significantly more suppressive of T cell activation relative to normal controls. The granulocytic but not monocyte subset of MDSCs (distinguished by CD15 expression) were identified as the main responders to PMA stimulation. Our results demonstrate immune suppression in newly diagnosed invasive breast cancer is characterized by increased immune suppressive function of MDSCs in the absence of increased cellular levels. When we looked at our cases with the highest MDSC levels, no distinct trend towards advanced stage or poorer prognosis biologic subtype was identified. These data support the identification of MDSCs in breast cancer as a novel therapeutic target to improve treatment outcomes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5405. doi:1538-7445.AM2012-5405

Tumor‐induced changes in the phenotype of blood‐derived and tumor‐associated T cells of early stage breast cancer patients

Tumor-induced immune suppression has mainly been studied in patients with advanced cancer. Despite the fact that they are most likely to benefit from immunotherapy, patients with early stage cancers were under-represented in these studies. We analyzed blood and tumor-derived T cells from patients with stage 1 (n = 20), stage 2 (n = 23) or stage 3 (n = 1) breast cancer and found that, even early stage tumors induced T cell differentiation. Breast cancer patients had significantly more circulating CD8+ memory and fewer CD8+ naïve T cells than healthy controls (n = 10). Up-regulation of CD69 and PD1 on cancer patient T cells suggests previous activation, and increased expression of the chemokine receptors CCR5 and CXCR3 on CD8+ T cells indicates that their homing capacity differs from that of healthy individuals. Comparison of blood-derived and tumor-associated T cells from patients with different metastatic status and tumor grades revealed that tumor progression and aggressiveness seem to favor the expansion of memory T cells over naive T cells. We have previously shown that immunosuppression in this patient population is stronger in the tumor than in the blood. Here, we report signs of exhaustion, such as loss of CD28, on tumor-associated as compared to blood-derived CD8+ T cells, despite the fact that tumor-associated T cells are predominantly effector memory cells and express high levels of CD69. The finding that the presence of a tumor potentially induces immunosenescence early during tumorigenesis indicates that efficient immunotherapy might be difficult even in patients with early stage cancer due to T cell exhaustion and tolerance.

Tumor Escape and Progression under Immune Pressure

Tumor Escape and Progression under Immune Pressure

Recent Advances in Cancer Immunotherapy

Cancer immunotherapy is designed to activate and up-regulate the host immune responses against tumor cells and is currently in use or under investigation for cancer treatments. Although tumor cells can use a variety of mechanisms to create an immunosuppressive environment, many agents and strategies to reduce or eliminate tumor-induced immune suppression are either being developed or already in clinical trials. This review deals with recent advances in cancer immunotherapy, focusing on new strategies to enhance antitumor immunity. Numerous approaches have been tested for the development of therapeutic cancer vaccines. Recently, several promising reports suggest that clinical benefit has been shown in randomized phase III trials for vaccines against prostate cancer, follicular lymphoma, and melanoma. Moreover, in 2010, the first patient-specific immunotherapy (sipuleucel-T) was approved by the FDA for the treatment of prostate cancer. On the other hand, some chemotherapeutic agents such as cyclophosphamide and docetaxel, have direct effects on the immune system that is likely to contribute to an improved antitumor immunity. Therefore, cancer immunotherapy combined with chemotherapy may represent one approach for breaking tumor-induced immune suppression. To date, a number of monoclonal antibodies are in use or under evaluation. Among these antibodies, blocking negative immunoregulatory receptors, CTLA-4 and PD-1, also represent a new strategy to induce antitumor immunity by manipulation of the immune system. In clinical trials, these antibodies could increase anti-tumor T-cell immunity and result in objective tumor regression in some patients. Thus, accumulating data from recent cancer vaccine trials, the manipulation of immune suppression using chemotherapeutic agents, and the use of immune-stimulatory antibodies raise the hope that immunotherapy could become the fourth modality of cancer treatment. Based on these new findings, in our hospital, a clinical trial using dendritic cells combined with low dose cyclophosphamide and docetaxel in patients with relapsed and refractory head and neck squamous cell carcinoma is under way (UMI03725, ClinicalTrials.gov identifier, NCT01149902). In the near future, various immunotherapeutic modalities will most probably be applied as novel approaches in the treatment of head and neck cancer.

The Role of the Adenosinergic Pathway in Immunosuppression Mediated by Human Regulatory T Cells (Treg)

Tumor-induced dysfunction of immune cells is a common problem in cancer. Tumors induce immune suppression by many different mechanisms, including accumulation of regulatory T cells (Treg). Adaptive Treg (Tr1) generated in the tumor microenvironment express CD39 and CD73 ectonucleotidases, produce adenosine and are COX2+PGE2+. Adenosine and PGE2 produced by Tr1 or tumor cells bind to their respective receptors on the surface of T effector cells (Teff) and cooperate in up-regulating cytosolic 3'5'-cAMP levels utilizing adenylyl cyclase isoform 7 (AC-7). In Teff, increased cAMP mediates suppression of anti-tumor functions. Treg, in contrast to Teff, seem to require high cAMP levels for mediating suppression. This differential requirement of Treg and Teff for cAMP offers an opportunity for pharmacologic interventions using selected inhibitors of the adenosine/PGE2 pathways. Blocking of adenosine/PGE2 production by Tr1 or blocking binding of these factors to their receptors on T cells or inhibition of cAMP synthesis in Teff all represent novel therapeutic strategies that used in combination with conventional therapies could restore anti-tumor functions of Teff . At the same time, these inhibitors could disarm Tr1 cells by depriving them of the factors promoting their generation and activity or by down-regulating 3'5'-cAMP levels. Thus, the pharmacologic control of Treg-Teff interactions offers a novel strategy for restoration of anti-tumor Teff functions and silencing of Treg. Used in conjunction with anti-cancer drugs or with immune therapies, this strategy has a potential to improve therapeutic effects by preventing or reversing tumor-induced immune suppression.

Potent Immunomodulatory Effects of Lenalidomide on Effector T Cells and Treg Improve the Effectiveness of a Therapeutic Lymphoma Vaccine

Abstract 108Lenalidomide is an effective therapeutic agent with direct inhibitory effects on malignant B- and plasma cells and immunomodulatory effects on the tumor microenvironment. The dual functions of Lenalidomide make it an appealing candidate for combination with other novel agents for lymphoma and myeloma therapy. In this study, we investigated the immune stimulatory effects of Lenalidomide on the potency of a model lymphoma vaccine (MCP3-sFv). Therapeutic vaccines based on the idiotype of the clonal Ig receptor on malignant B cells have been shown previously to elicit specific immunity in human patients, and a recent controlled Phase III trial of an idiotype protein vaccine demonstrated prolongation of disease free survival in follicular lymphoma patients (J Clin Oncol. 10;29(20):2787–94 2011). In prophylactic experiments, the groups of 10 BALB/c mice immunized with a novel fusion DNA idiotype vaccine (MCP3-sFv, Science. 1;298(5595):1025–9 2002) were then treated with Lenalidomide at various doses (5–50 mg/kg) or saline as control i.p. for 35 consecutive days. This combination strategy protected mice from a lethal syngeneic A20 murine lymphoma, resulting in significantly improved survival comparing with vaccine or Lenalidomide alone or saline. Furthermore, more than 70% of surviving mice treated with the combination were resistant to tumor re-challenge suggesting memory antitumor immunity. Mechanistically, protection required effector cellular immunity, as tumor protection was abrogated by treatment of depleting antibody against CD8+ T cells, either alone or together with CD4 depleting antibody in vivo. Lenalidomide showed little impact on humoral immunity, with serum anti-idiotype antibody levels in the combination group similar to the vaccine alone group. In addition to adaptive immune system, we also examined the effect of Lenalidomide on other immune cells including NK cells, myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg). Interestingly, Lenalidomide treatment did not affect the number of these immune cell populations in non-tumor bearing mice; but in tumor-bearing mice, Lenalidomide-induced tumor regression was associated with reduced numbers of immune suppressive cells (MDSC/Treg) and rescue of circulating NK cells. This finding suggests a role of Lenalidomide in ameliorating tumor-induced immune suppression. This hypothesis was supported by the demonstration that the combination of Lenalidomide and vaccine (vaccine given starting on day 8) produced significantly improve survival, compared with controls receiving vaccine or Lenalidomide alone (log rank p<0.05). In conclusion, the opposing effects of Lenalidomide on enhancing cellular immunity and ameliorating immune suppression make it ideal for combination with active specific immunotherapy for treatment of hematological malignancies. Disclosures:Kwak:Biovest International, Antigenics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biovest International: ; Antigenics, Xeme Biopharma: Equity Ownership.

Decreased accumulation of immune regulatory cells is correlated to the antitumor effect of IFN-γ overexpression in the tumor

During mammary tumorigenesis, there is a profound tumor-induced immunosuppression and a progressive thymic atrophy associated with tumor development. IFN-γ has been shown to be effective in enhancing antitumor responses in several tumor models, however, how IFN-γ exerts its anti-tumor effect is largely controversial. In the present study we have used a mammary tumor model to investigate whether the levels of IFN-γ have an important role in the tumor-induced immuno-suppression as well as in the pathogenesis of the thymic atrophy. We evaluated this possibility using DA-3 cells transfected to express IFN-γ (DA-3/IFN-γ), a system that provides constant, local production of IFN-γ within the tumor microenvironment. Overexpression of IFN-γ in the mammary tumor results in a marked delay of tumor growth, a reduction in regulatory T cells and myeloid-derived suppressor cells accumulation mostly due to down-regulation of chemokines implicated in the recruitment of immune regulatory cells, and a blockage in the tumor-associated thymus atrophy. Collectively, our data suggest that the replacement of the faulty levels of IFN-γ in the tumor results in a diminution of the tumor-induced immune suppression caused by the mammary tumor development.