Cell Antitumor Immune Responses Research Articles

Advances of therapeutic cancer vaccines

Therapeutic cancer vaccines offer a novel avenue for the development of cancer immunotherapy. Tumor antigens can induce T cell antitumor immune responses, but lack of tumor-specific antigens has hampered clinical efficacy. Vaccine types include peptide vaccines, dendritic cell vaccines and oncolytic virus vaccines. Personalizing tumor vaccines may provide a new immunotherapeutic strategy to treat cancer. Key words: Neoplasms; Immunotherapy; Cancer vaccines

Enhanced immunotherapy by combining a vaccine with a novel murine GITR ligand fusion protein

Immunotherapy was significantly enhanced in a murine tumor model by combining a vaccine with a fusion protein designed to target the glucocorticoid-induced tumor necrosis factor (TNF) receptor related gene (GITR) on the surface of T cells. The recombinant poxvirus-based vaccine platform included Modified Vaccinia virus Ankara (rMVA) and fowlpox (rF) vectors as the driver immunogens both engineered to express the human carcinoembryonic antigen (CEA) and three murine costimulatory molecules B7.1, ICAM-1, LFA-3 (designated TRICOM). In previous studies, mice expressing human CEA as a transgene (CEA.Tg mice) vaccinated with rMVA/rF-CEA-TRICOM overcame CEA immune tolerance by inducing anti-CEA‒specific immunity and regression of CEA-expressing tumors. The murine GITR ligand fusion protein (mGITRL-FP) consisted of a mouse IgG2a Fc region, a yeast-derived coiled GCN4 pII and the extracellular GITR-binding domain of murine GITR ligand. The design maximized valency and the potential to agonize the GITR receptor. Combined treatment of the vaccine and mGITRL-FP mediated a more robust tumor regression, leading to sustained improvement in overall survival. The enhanced immunotherapeutic effect was linked to the generation of a strong CD8+ T cell antitumor immune response. A treatment schedule with mGITRL-FP administered prior to the priming rMVA-CEA-TRICOM vaccination was of paramount importance. The mechanism of action for the enhanced antitumor effects resided in the depletion of immune cells, particularly FoxP3+ regulatory T cells, that express high GITR levels following activation. The results provide evidence that targeting GITR with mGITRL-FP in concert with a cancer vaccine represents a potential novel approach to more effective immunotherapy.

Chemotherapy and tumor immunogenic cell death

Chemotherapy resist is the problem for clinic, and some researches find that chemotherapy with anthracycline and oxaliplatin not only induces the tumor cell apoptosis, but also the cell immunogenic cell death (ICD) by inducing the tumor cell apoptosis and releasing three kinds of signals: exposure of calreticulin on the cell surface to stimulate the dendritic cell (DC) to engulf, and the secretion of adenosine triphosphate to recruit DC to enter into tumor bed, and the release of the high mobility group B1 to promote DC to steadily bind with dying tumor cell to induce specific T cell antitumor immune response. It is with great meaning to promote the chemotherapy protocol by studying the ICD induced by chemotherapy. Key words: Drug therapy; T-cell antigen receptor specificity; Dendritic cells; Calreticulin; Immunogenic cell death

LYG1 exerts antitumor function through promoting the activation, proliferation, and function of CD4+ T cells

ABSTRACTIdentification of novel stimulatory cytokines with antitumor function would have great value in tumor immunotherapy investigations. Here, we report LYG1 (Lysozyme G-like 1) identified through the strategy of Immunogenomics as a novel classical secretory protein with tumor-inhibiting function. LYG1 recombinant protein (rhLYG1) could significantly suppress the growth of B16 tumors in WT B6 mice, but not in SCID-beige mice, Rag1−/− mice, CD4+- or CD8+ T cell-deleted mice. It could increase the number of CD4+ and CD8+ T cells in tumor-infiltrating lymphocytes, tumor-draining lymph nodes, and spleens, and promote IFNγ production by T cells in tumor-bearing mice. In vitro experiments demonstrated that rhLYG1 could directly enhance IFNγ secretion by CD4+ T cells, but not CD8+ T cells. Moreover, it could promote the activation, proliferation, and IFNγ production of tumor antigen-specific CD4+ T cells. The tumor-inhibiting effect of LYG1 was eliminated in Ifng−/− mice. Furthermore, LYG1 deficiency accelerated B16 and LLC1 tumor growth and inhibited the function of T cells. In summary, our findings reveal a tumor-inhibiting role for LYG1 through promoting the activation, proliferation, and function of CD4+ T cells in antitumor immune responses, offering implications for novel tumor immunotherapy.

Chromosome 9p24.1/PD-L1/PD-L2Alterations and PD-L1 Expression and Treatment Outcomes in Patients with Classical Hodgkin Lymphoma Treated with Nivolumab (PD-1 Blockade)

Chromosome 9p24.1/PD-L1/PD-L2Alterations and PD-L1 Expression and Treatment Outcomes in Patients with Classical Hodgkin Lymphoma Treated with Nivolumab (PD-1 Blockade)

RPN13/ADRM1 inhibitor reverses immunosuppression by myeloid-derived suppressor cells.

Myeloid-derived-suppressor cells (MDSCs) are key mediators of immune suppression in the ovarian tumor microenvironment. Modulation of MDSC function to relieve immunosuppression may enhance the immunologic clearance of tumors. The bis-benzylidine piperidone RA190 binds to the ubiquitin receptor RPN13/ADRM1 on the 19S regulatory particle of the proteasome and directly kills ovarian cancer cells by triggering proteotoxic stress. Here we examine the effect of RA190 treatment on the immunosuppression induced by MDSCs in the tumor microenvironment, specifically on the immunosuppression induced by MDSCs. We show that RA190 reduces the expression of Stat3 and the levels of key immunosuppressive enzymes and cytokines arginase, iNOS, and IL-10 in MDSCs, while boosting expression of the immunostimulatory cytokine IL-12. Furthermore, we show that the RA190-treated MDSCs lost their capacity to suppress CD8+ T cell function. Finally, we show that RA190 treatment of mice bearing syngeneic ovarian tumor elicits potent CD8+ T cell antitumor immune responses and improves tumor control and survival. These data suggest the potential of RA190 for ovarian cancer treatment by both direct killing of tumor cells via proteasome inhibition and relief of MDSC-mediated suppression of CD8 T cell-dependent antitumor immunity elicited by the apoptotic tumor cells.

Abstract 4977: Immunogenic chemotherapy synergize PD-1 blockade by enhancing dendritic cells infiltration in triple-negative breast cancer (TNBC)

Abstract Targeting immune checkpoints such as programmed cell death protein 1 (PD1), programmed cell death 1 ligand 1 (PDL1) has achieved noteworthy benefit in multiple cancers. However, their efficacy remains unsatisfactory in triple-negative breast cancer (TNBC). Breast cancer is not highly mutagenic and immune checkpoints blockade therapies for TNBC is not as effective as for melanoma or lung cancer with high mutation rates. Combination of different modalities offers an opportunity to enhance their effect. We studied the synergistic effect of cancer chemotherapy and immunotherapy in TNBC mouse model. Here we report that the combination of Doxisome (liposomal encapsulated formulation of Doxorubicin) and anti-PD-1 antibody suppresses tumor growth, reduces lung metastasis and improves survival of mice bearing 4T1-triple negative breast cancer. Moreover, the combination of Doxisome and anti-PD-1 was able to overcome low dose Doxisome resistance in mouse models of TNBC. The success of anticancer chemotherapy is linked to a durable immune response of targeting tumor. We present evidence that the synergistic therapeutic activity of Doxisome with anti-PD1 blockade results in enhanced T cell anti-tumor immune responses. Moreover, the synergetic effect is due to increased dendritic cells (DCs) infiltration in tumor microenvironment, which internalize tumor antigens and subsequently activate tumor-reactive T cells. In TNBC mouse model, by combining anti-PD1 checkpoint blockade while shifting the immune response towards CD4+ Th1 with Doxisome, immune tolerance to TNBC tumor can be overcome. Enhancing antigen presenting cells (APCs, e.g. DCs) infiltration by immunogenic chemotherapy and T cell activation by anti-PD1 blockade increase therapy response in TNBC. These results provide rationale for several newly planned clinical trials combining immunogenic chemotherapy with PD-1 or PD-L1 blockade in breast cancer and exploration of such a combination in others solid tumors. Citation Format: Xiangliang Yuan, Yi Xiao, Wenling Kuo, Dihua Yu. Immunogenic chemotherapy synergize PD-1 blockade by enhancing dendritic cells infiltration in triple-negative breast cancer (TNBC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4977.

Abstract LB-089: Imprime PGG, a β-glucan PAMP (pathogen-associated molecular pattern), effectively elicits in vivo maturation of antigen presenting cells in mice and humans, suggesting potential synergy with checkpoint inhibitor therapy

Abstract Recognition of PAMPs via pattern recognition receptors is central to immune recognition of foreign threats and to the generation of a coordinated innate and adaptive immune response. Cancers lack PAMPs and are poorly immunogenic. Consequently, the immune system often fails to mount an effective, coordinated anti-cancer immune attack. Though immunotherapies (e.g. checkpoint inhibitors) are effective in some cancer patients, the majority of patients fail to achieve substantial therapeutic benefit. To fully realize the potential of immune checkpoint inhibitors, there is substantial interest in developing therapeutically-viable PAMPs capable of enabling the maturation and function of professional antigen presenting cells (e.g. dendritic cells, DCs). Bacterial and viral PAMPs (i.e. TLR and STING agonists) can elicit DC maturation but are poorly tolerated systemically and are thereby limited to intra-tumoral delivery. The soluble yeast β-1,3/1,6 glucan, Imprime PGG (Imprime), is a PAMP that has been successfully administered intravenously (IV), is well-tolerated and shows promising efficacy in a series of clinical trials in > 400 total subjects. We sought to determine whether Imprime could drive maturation and enhanced function of antigen presenting cells in vivo. We now show that Imprime binds in vivo to various dendritic cell (DC) subsets in both human and mouse. In mice dosed IV, Imprime also elicits DC maturation as indicated by CD86 upregulation and successfully induces a type I interferon transcriptional profile. In C57Bl/6 mice immunized with the OVA257-264 model antigen, Imprime treatment elicits the specific expansion of adoptively transferred OT-I CD8 T cells (transgenic T cells engineered to recognize the OVA antigen). These OT-I T cells are functional effector cells, showing enhanced degranulation and increased capacity to produce IFN-γ and IL-2 when compared to OT-I cells isolated from mice challenged with OVA peptide alone. In ex vivo human whole blood, Imprime also enables DC maturation (enhanced expression of CD86, CD83, MHC class II), T cell expansion and production of the potent anti-tumor cytokine IFN-γ. Importantly, we now show preliminary data that peripheral blood monocytes and classical DCs from Imprime-treated cancer patients show elevated CD86 expression. Collectively, these data show that Imprime, a novel, systemically-administered, well-tolerated PAMP, can effectively elicit the maturation and function of antigen presenting cells in vivo and may thereby enhance T cell priming and anti-tumor immune response elicited by checkpoint inhibitors. Citation Format: Ross B. Fulton, Steven M. Leonardo, Adria B. Jonas, Kathryn A. Fraser, Anissa S.H. Chan, Nadine R. Ottoson, Michael E. Danielson, Nandita Bose, Jeremy R. Graff, Keith Gorden. Imprime PGG, a β-glucan PAMP (pathogen-associated molecular pattern), effectively elicits in vivo maturation of antigen presenting cells in mice and humans, suggesting potential synergy with checkpoint inhibitor therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-089.

Pro- and anti-tumour effects of B cells and antibodies in cancer: a comparison of clinical studies and preclinical models.

The primary immune role of B cells is to produce antibodies, but they can also influence T cell function via antigen presentation and, in some contexts, immune regulation. Whether their roles in tumour immunity are similar to those in other chronic immune responses such as autoimmunity and chronic infection, where both pro- and anti-inflammatory roles have been described, remains controversial. Many studies have aimed to define the role of B cells in antitumor immune responses, but despite this considerable body of work, it is not yet possible to predict how they will affect immunity to any given tumour. In many human cancers, the presence of tumour-infiltrating B cells and tumour-reactive antibodies correlates with extended patient survival, and this clinical observation is supported by data from some animal models. On the other hand, T cell responses can be adversely affected by B cell production of immunoregulatory cytokines, a phenomenon that has been demonstrated in humans and in animal models. The isotype and concentration of tumour-reactive antibodies may also influence tumour progression. Recruitment of B cells into tumours may directly reflect the subtype and strength of the anti-tumour T cell response. As the response becomes chronic, B cells may attenuate T cell responses in an attempt to decrease host damage, similar to their described role in chronic infection and autoimmunity. Understanding how B cell responses in cancer are related to the effectiveness of the overall anti-tumour response is likely to aid in the development of new therapeutic interventions against cancer.

The role of stromal cells in anti-tumor immune responses

Pancreaticductal adenocarcinoma (PDAC) is a cancer of the exocrine pancreas with an aggressive prognosis (a 5-year survival rate of 6 months), resulting in more than 250,000 deaths per year. PDAC is the fourth most common cause of cancer-related mortality worldwide. PDAC is more resistant to chemotherapy than other types of cancer due to the dense fibrosis around it. Moreover, only 15% of patients who have PDAC tumors can have surgical resection. Consequently, the success of chemotherapy and surgery in PDAC treatments is limited, motivating researchers to turn to immunotherapy modalities. The dense fibrotic nature of the PDAC microenvironment makes it worthwhile to research the cancer-associated fibroblasts (CAF). The CAF cells play a role in the formation of fibrotic barriers, the altering of the anti-tumor immune response, and supporting to carcinogenesis. Many immune system elements, such as macrophages and cytotoxic T cells, play a role in the formation of the anti-tumor immune response. Macrophages play a particularly crucial role in that anti-tumor immune response but in two opposing manners, activating classically or in alternative ways. Macrophages that activate in alternative manners, the tumor associated macrophages (TAMs), have become a focus in cancer research in recent years due to their role in suppressing the anti-tumor response, in supporting angiogenesis and metastasis. However, there is no consensus on how the TAM cells are polarized from monocytes. This study aims to determine whether or not CAF cells play a role in the polarization of TAMs through CXCL12/ CXCR4 axis, and how this role affects survival and tumor volume in PDAC patients. If we explore that CAFs play a role in macrophage polarization viaCXCL12 / CXCR4 axis in this study, we may suppress this pathway and gain clinical benefit from this inhibition in PDAC patients.

Detection of antibody therapy-induced anti-tumor immune responses using anti-CD8 immuno-pet

Tumor heterogeneity and the dynamic tumor immune microenvironment have become important topics in the field of cancer immunotherapy. The ability to noninvasively monitor immune cells in vivo via surface markers on immune cell subsets using immuno-positron emission tomography (immuno-PET) is an attractive means of visualizing both systemic and intratumoral alterations in immune cell numbers and localization during experimental immunotherapies. Due to the critical role of tumor-infiltrating cytotoxic CD8+ T cells in anti-tumor immune responses, a radiolabeled anti-CD8 antibody fragment (cys-diabody) was developed for immuno-PET detection of cytotoxic CD8+ T cells in vivo. The anti-CD8 cys-diabody radiolabeled with 89Zr using the bifunctional chelator deferoxamine-maleimide enabled visualization of the spleen and lymph nodes in normal mice; specificity was confirmed in CD8-blocking studies. Next, anti-CD8 immuno-PET was shown to specifically detect tumor-infiltrating lymphocytes in immunotherapy models including agonistic antibody therapy (anti-CD137/4-1BB) and checkpoint blockade antibody therapy (anti-PD-L1). Balb/c mice bearing week old syngeneic CT26 colorectal tumors were treated with either anti-CD137 or anti-PD-L1 antibody therapy and imaged at day 8 post-therapy. All mice treated with anti-CD137 therapy demonstrated tumor regression and increased anti-CD8 immuno-PET targeting in the tumors of mice treated with anti-CD137 therapy compared to the tumor of untreated mice. Ex vivo biodistribution confirmed enhanced tumor uptake of anti-CD137 treated versus untreated mice (15 ± 5.5 %ID/g versus 6.0 ± 0.83 %ID/g, respectively). Anti-CD8 immuno-PET of untreated mice demonstrated a rim of activity around the tumor while anti-CD137 treated mice demonstrated enhanced intratumoral uptake. The increase in CD8+ tumor-infiltrating lymphocytes was validated by both flow cytometry and immunohistochemistry. About 30% of mice treated with anti-PD-L1 therapy demonstrated tumor regression and were grouped into non-responders (average tumor diameter > 8mm) and responders (average tumor diameter < 8mm). Anti-PD-L1 non-responders and responders had tumor anti-CD8 immuno-PET uptake of 6.4 ± 1.5 %ID/g and 11.27 ± 2.7 %ID/g as determined from ex vivo biodistribution analysis, respectively, demonstrating enhanced CD8+ T cell infiltration in responding mice that was confirmed by flow cytometry. Anti-CD8 immuno-PET was successful in the noninvasive detection of tumor-infiltrating CD8+ T cells in two differing antibody-based immunotherapy strategies that have shown promise in the clinic.

Abstract 251: Combining Ibrutinib with immune checkpoint blockade to induce therapeutic antitumor immune response in solid tumors

Abstract The ability of T-cell responses to effectively attack tumors is restricted by multiple negative checkpoint regulators. Monoclonal antibodies blocking negative regulators of T -cells, such as CTLA-4 and PD-L1 can amplify pre-existing immune responses and evoke new therapeutic responses. Ibrutinib (Imbruvica®), a covalent inhibitor of BTK has shown therapeutic efficacy in several blood cancers, by a mechanism thought to be a direct effect on a critical signaling pathway intrinsic to the malignant B cell. Since BTK is predominantly expressed in hematopoietic cells, solid tumors have not been an obvious target of this drug. An additional target of ibrutinib is the inhibition of ITK, a key enzyme in the survival of Th2 T cells. It has been reported that ibrutinib can alter the balance of Th1 and Th2 T cells leading to enhanced immune responses. Here we report that the combination of ibrutinib and a-PD-L1 antibody suppresses tumor growth, reduces lung metastasis and improves survival of mice bearing solid tumors such as 4T1-triple negative breast cancer and CT26-colon cancer. Moreover, the combination of ibrutinib and a-PD-L1 was able to overcome ibrutinib resistance in mouse models of lymphoma and myeloma. We present evidence that the synergistic therapeutic activity of Ibrutinib with anti PD1 blockade results in enhanced T cell anti-tumor immune responses. In preclinical models, by combining inhibitory checkpoint blockade while shifting the immune response towards CD4 Th1 with ibrutinib, immune tolerance to tumors can be overcome. These results provide rationale for several newly planned clinical trials combining ibrutinib with PD-1 blockade in solid tumor and exploration of such a combination in hematologic malignancies. Citation Format: Idit Sagiv-Barfi, Holbrook Kohrt, Debra Czerwinski, Patrick Ng, Betty Chang, Ronald Levy. Combining Ibrutinib with immune checkpoint blockade to induce therapeutic antitumor immune response in solid tumors. [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 251. doi:10.1158/1538-7445.AM2015-251

Abstract IA22: Innate immune cell PI3K gamma as a target for suppression of pancreatic ductal adenocarcinoma

Abstract Ductal adenocarcinoma of the pancreas (PDAC) is a devastating disease that will afflict greater than 40,000 Americans. Less than 5% of these new pancreatic cancer patients will survive 5 years following diagnosis. PDAC is rarely detected in the early stages, with more than 80% of patients presenting with locally unresectable or metastatic disease at the time of diagnosis. As this form of cancer is also resistant to current cytotoxic therapies and ionizing radiation, novel therapeutic and diagnostic approaches are desperately needed to improve patient outcome. A characteristic feature of PDAC is the presence of an abundant, inflammatory infiltrate. In response to tumor-derived chemoattractants, we found that myeloid cells invade tumors where they promote immunosuppression, resulting in tumor growth and metastasis. We discovered that myeloid cell PI3-kinase gamma (PI3Kgamma) controls tumor immunosuppression, as PI3Kgamma signaling inhibits pro-inflammatory gene expression responses in macrophages, monocytes and granulocytes and promotes anti-inflammatory responses. Suppression of PI3Kgamma in mutant mice and in mice treated with pharmacologic inhibitors of PI3Kgamma promotes CD8+ T cell anti-tumor immune responses, leading to a 50% reduction in tumor growth and metastasis in orthotopic mouse models of pancreatic carcinoma and significant extension of survival in GEM models of PDAC (PDX1-cre; LSL-KrasG12D/+; LSL-Trp53 R172H/+). Furthermore, PI3Kgamma inhibition blocks macrophage stimulation of collagen deposition by fibroblasts in PDAC. We are exploring the combination of PI3kinase gamma inhibition with tumor cell and T cell targeted therapeutics could significantly reduce tumor growth and metastasis of PDAC. These approaches could improve patient outcomes by reducing tumor progression and preserving organ function by inhibiting desmoplasia. Citation Format: Megan Kaneda, Chanae Hardamon, Michael C. Schmid, Michael Bouvet, Franco Novelli, Emilio Hirsch, Andrew Lowy, Judith A. Varner. Innate immune cell PI3K gamma as a target for suppression of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr IA22.

Chronic alcohol consumption inhibits melanoma growth but decreases the survival of mice immunized with tumor cell lysate and boosted with α-galactosylceramide

Alcohol consumption increases the incidence of multiple types of cancer. However, how chronic alcohol consumption affects tumor progression and host survival remains largely unexplored. Using a mouse B16BL6 melanoma model, we studied the effects of chronic alcohol consumption on s.c. tumor growth, iNKT cell antitumor immune response, and host survival. The results indicate that although chronic alcohol consumption inhibits melanoma growth, this does not translate into increased host survival. Immunizing mice with a melanoma cell lysate does not significantly increase the median survival of water-drinking, melanoma-bearing mice, but significantly increases the median survival of alcohol-consuming, melanoma-bearing mice. Even though survival is extended in the alcohol-consuming mice after immunization, the median survival is not different from the immunized mice in the water-drinking group. Immunization with tumor cell lysate combined with α-galatosylceramide activation of iNKT cells significantly increases host survival of both groups of melanoma-bearing mice compared to their respective non-immunized counterparts; however, the median survival of the alcohol-consuming group is significantly lower than that of the water-drinking group. Alcohol consumption increases NKT cells in the thymus and blood and skews NKT cell cytokine profile from Th1 dominant to Th2 dominant in the tumor-bearing mice. In summary, these results indicate that chronic alcohol consumption activates the immune system, which leads to the inhibition of s.c. melanoma growth and enhances the immune response to immunization with melanoma lysate. With tumor progression, alcohol consumption accelerates iNKT cell dysfunction and compromises antitumor immunity, which leads to decreased survival of melanoma-bearing mice.

SUMO2 overexpression enhances the generation and function of interleukin-17-producing CD8+ T cells in mice

Small ubiquitin-like modifier (SUMO) 2 is a small protein that controls the activity and stability of other proteins by SUMOylation. In this study, T cell-specific SUMO2 overexpressing transgenic mice were generated to study the effect of SUMO2 on T lymphocytes. SUMO2 overexpression promoted differentiation of interleukin (IL)-17-producing CD8+ T cells, and significantly suppressed the growth of EL4 tumor cells in vivo. Moreover, the tumor tissue from SUMO2-overexpressing mice had higher interferon (IFN)-γ and granzyme B mRNA levels. Although SUMO2 overexpression did not increase IFN-γ or granzyme B production in cytotoxic T lymphocytes, IL-12 treatment restored and increased IFN-γ secretion in IL-17-producing CD8+ T cells. SUMO2 overexpression also increased gene expression of chemokines, CCL4, and CXCL10, which attract cytotoxic T lymphocytes to tumor tissues. Additionally, SUMO2-overexpressing T cells exhibited increased STAT3 phosphorylation, implying a SUMO2 target which up-regulates STAT3 activity governing IL-17A-producing CD8+ T cell differentiation and antitumor immune responses.

CUL4A contributes to the biology of basal-like breast tumors through modulation of cell growth and antitumor immune response.

The CUL4A E3 ubiquitin ligase is involved in the regulation of many cellular processes and its amplification and/or overexpression has been observed in breast cancer. The 13q34 amplification, which is associated with the basal-like breast cancer subtype, has been proposed as one of the mechanism behind CUL4A up-regulation. However, the specific contribution of CUL4A to the biology of basal-like breast tumors has not yet been elucidated. In this work, by using cellular models of basal phenotype, we show the inhibitory effect of CUL4A silencing in the proliferation and growth of breast cancer cells both, in vitro and in vivo. We also demonstrate the transforming capacity of CUL4A exogenous overexpression in the 184B5 human mammary epithelial cells in vitro. Our results suggest a synergistic effect between CUL4A high levels and the activation of the RAS pathway in the tumorigenesis of basal-like breast cancer tumors. In addition, by using a proteomics approach we have defined novel candidate proteins and pathways that might mediate the oncogenic effect of CUL4A. In particular, we report a putative role of CUL4A in bypassing the immune system in breast cancer through the down-regulation of several molecules involved in the immune surveillance. These findings provide insight into the oncogenic properties of CUL4A in basal-like breast cancer and highlight the therapeutic opportunities to target CUL4A.

Hepatocellular Carcinoma from an Immunologic Perspective

Hepatocellular carcinoma is the third most common cancer worldwide. It is an inflammation-associated cancer. Multiple investigators have demonstrated that analysis of the tumor microenvironment may be used to predict patient outcome, indicating the importance of local immune responses in this disease. In contrast with other types of cancer, in which surgery, radiation, and systemic cytotoxic chemotherapies dominate the treatment options, in hepatocellular carcinoma locoregional treatments are widely applied. Such treatments induce rapid tumor cell death and antitumor immune responses, which may favor or impair the patients' outcome. Recent immunotherapeutic studies demonstrating promising results include trials evaluating intratumoral injection of an oncolytic virus expressing granulocyte macrophage colony-stimulating factor, glypican-3 targeting treatments, and anti-CTLA4 treatment. Although some of these novel approaches may provide benefit as single agents, there is a clear opportunity in hepatocellular carcinoma to evaluate these in combination with the standard modalities to more effectively harness the immune response.

Different immunohistochemical levels of Hsp60 and Hsp70 in a subset of brain tumors and putative role of Hsp60 in neuroepithelial tumorigenesis

In this work we analysed, by immunohistochemistry, a series of brain tumors to detect the levels and cellular distribution of Hsp60 and Hsp70. We found that Hsp60 levels were significantly higher than those of Hsp70 in neuroepithelial tumors, while levels of both molecules were not significantly different from each other in meningeal neoplasms. In particular, Hsp60 immunopositivity was present mainly at the cytoplasmic level, while Hsp70 immunopositivity was found both in the cytoplasm and in the nucleus of tumor cells. The levels of these molecules in healthy control cells were always very low. Finally, Hsp60 and Hsp70 levels did not correlate with the different types (WHO grade) of neoplasm. Our results are partially in agreement with previous studies and suggest that Hsp60 is not increased by a passive phenomenon (e.g., due to the stress caused by the peritumor environment on cancer cells) but may be actively implicated in tumor progression, e.g. inhibiting tumor cell death or antitumor immune system response, as already postulated in vitro. We also briefly discuss the most recent publications on the extramitochondrial localization of Hsp60 in tumor cells and its role in tumor progression.

Abstract 4975: The role of the Ikaros transcription factor in regulatory T cell (Treg) development and function in a murine pancreatic adenocarcinoma model.

Abstract Pancreatic cancer (PC) is currently the only major form of cancer that still has survival rates in the single digits. The limited success of cancer therapies in treating pancreatic cancer has been primarily due to the immunosuppressive tumor microenvironment. In particular, there is an increased prevalence of regulatory T cells (Tregs) (CD4+CD25+CD127−), which suppress anti-tumor immune responses in PC tumor-bearing (TB) hosts. Tregs express the Forkhead BoxP3 (FoxP3) gene, which is critical for their suppressive function. Studies have shown that Ikaros, a zinc finger transcription factor, is an important regulator of T lymphocyte development and function. The regulation of Ikaros’ expression and function is controlled by post-translational modification events. Deficiencies in Ikaros are observed in various T cell leukemias and lymphomas. However, little is known about the possible role of Ikaros in regulating immune cell development in response to solid cancers. In this study, we aim to identify the role of Ikaros in regulating Treg homeostasis and function in a pancreatic tumor microenvironment. Therefore, using our murine model of pancreatic cancer, we isolated splenocytes from TB and control mice and performed flow cytometry and magnetic activated cell sorting (MACS) to immunophenotype and enrich T cell populations for in vivo and in vitro analyses. Also, we performed quantitative real-time PCR (qRT-PCR) and western blot analyses to evaluate Ikaros and FoxP3 mRNA and protein expression in whole and enriched CD3+ T cells from TB and control splenocytes. Our results showed that effector T cell percentages (CD4+ and CD8+) were significantly lower in splenocytes from TB mice compared to control. However, our results also showed a significant expansion of Tregs in splenocytes from TB mice compared to control. In addition, enriched TB Tregs (CD4+CD25+) suppressed antigen-specific CD8+T cell immune responses in a dose-dependent manner, in vitro. Preliminary qRT-PCR results revealed no significant difference in Ikaros mRNA expression; whereas, Ikaros protein expression was reduced in TB whole splenocytes compared to control. Also, Ikaros protein expression was reduced in TB enriched CD3+ T cells compared to control. Furthermore, our results showed an increase in FoxP3 protein expression in TB CD3+ T cells compared to control. These findings suggest that the pancreatic tumor microenvironment potentially down-regulates Ikaros’ protein expression, which may contribute to the expansion of Tregs and their suppression of CD8+T cell (anti-tumor) immune responses. Citation Format: Nadine Nelson, Maya Jerald, Laura Pendleton, Karoly Szekeres, Nasreen Vohra, Shari Pilon-Thomas, Tomar Ghansah. The role of the Ikaros transcription factor in regulatory T cell (Treg) development and function in a murine pancreatic adenocarcinoma model. [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 4975. doi:10.1158/1538-7445.AM2013-4975

The role of pro-inflammatory cytokines and immune cells in colorectal carcinoma progression

Cytokines exhibit a pleiotropic effect in the regulation of the immune cell function, tumor growth and antitumor immune responses. A total of 30 patients with colorectal carcinoma were enrolled on this study and their levels of interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, serum granulocyte colony-stimulating factor (sG-CSF) and serum macrophage colony-stimulating factor (sM-CSF) were measured preoperatively using ELISA. Tumor-infiltrating granulocyte (TIG), tumor-associated macrophage (TAM), G-CSF and M-CSF expression in tumor cells were examined using immunostaining. This study revealed abnormal levels of cytokines in patients, including IL-1β (1/30, 3.3%), IL-6 (16/30 53.3%), IL-81 (15/30, 50%), TNF-α (4/21, 19%), sG-CSF (17/30, 56.7%) and sM-CSF (4/21, 19%). There was a positive linear correlation between IL-6 and sM-CSF (P=0.017, R=0.517). sG-CSF was significantly associated with a deeper tumor invasion (P=0.039) and a more advanced tumor stage (P=0.023). The granulocyte/lymphocyte (G/L) ratio was associated with abnormal levels of sG-CSF. Logistic univariate analysis revealed that TIGs were a risk factor for lymph node metastasis (0.019) and TAMs were a risk factor for depth of invasion (0.029), but this was not confirmed in logistic multivariate analysis. In conclusion, IL-6, IL-8, sM-CSF and sG-CSF may indirectly promote tumor growth, progression and metastasis by changing the leukocyte populations in the blood and the tumor microenvironment.