bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B110: Proinflammatory allogeneic dendritic cells promote activation of bystander immune cells and indirectly license antigen-specific T-cells
Authors: Fotaki Grammatiki , Jin Chuan , Kerzeli Iliana Kyriaki , Ramachandran Mohanraj , Karlsson-Parra Alex , Yu Di , Essand Magnus
Published: February 2019

Autologous patient-derived dendritic cells (DCs) modified ex vivo to present tumor-associated antigens have frequently been utilized as cancer vaccines. However, apart from the stringent logistics in producing DCs on a patient basis, accumulating evidences indicate that such DCs are poor in migration and in fact do not directly interact with naïve T-cells in vivo. Instead, it is the bystander host-DCs taking up material from apoptotic vaccine DCs and subsequently initiate antitumor T-cell responses. We examine the possibility of harnessing allogeneic pro-inflammatory DCs (alloDCs) as immune enhancers which may affect the activation of bystander immune cells and promote antigen-specific T-cell responses. We have verified the inflammatory state of DCs matured with a maturation cocktail in combination with an infection-enhanced adenovirus. Such proinflammatory alloDCs expressed co-stimulatory and activation molecules and secreted Th-1 type cytokines in vitro. T and NK cells up-regulated activation markers after co-culture with such alloDCs and created an immunostimulatory environment which was found to promote maturation of antigen-loaded DCs and the subsequent activation of antigen-specific T-cells by cross-presentation. Our findings were evaluated as a therapeutic strategy in murine models with success. We are proposing that vaccination with proinflammatory alloDCs in combination with the use of Ad5M as possible antigen delivery vehicle can be a cost-effective strategy to induce antitumor immune responses.

Citation Format: Grammatiki Fotaki, Chuan Jin, Iliana Kyriaki Kerzeli, Mohanraj Ramachandran, Alex Karlsson-Parra, Di Yu, Magnus Essand. Proinflammatory allogeneic dendritic cells promote activation of bystander immune cells and indirectly license antigen-specific T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B110.
Abstract B112: Combined therapy with an autologous tumor cells/bacillus Calmette-Guérin/formalin-based vaccine plus anti-PD-1 inhibitor enhances the antitumor response in a 4T1 breast cancer model
Authors: Calderon Maria Jose Godoy , Marcano Eglys González , Convit Ana Federica
Published: February 2019

Immune checkpoint inhibitors have shown satisfactory results in different types of cancer, such as lung cancer and melanoma. However, in breast cancer their benefit as a stand-alone treatment have not been robust, suggesting the need of combined therapies. In preclinical and clinical studies some checkpoint inhibitors have shown enhancement of cancer vaccines’ antitumor effects. Cancer vaccines represent a promising immunotherapy since they induce a specific long-term immune response against tumor antigens, leading to an effective tumor elimination with minimal side effects. Based on successful previous experiences using an autologous tumor cells/bacillus Calmette-Guérin (BCG)/formalin-based vaccine in breast cancer, we aimed to enhance its antitumor properties by combining it with anti-PD-1. We used a 4T1 breast cancer model in BALB/c mice, receiving four weekly injections of either Phosphate Buffered Saline (G1), 50 μg anti-PD-1 (G2), or the vaccine plus anti-PD-1 (200 µg autologous tumor cells, 0.0625 mg BCG, 0.02% formalin plus 50 μg anti-PD-1) (G3). In G3, the tumor arrest ability of anti-PD-1 was enhanced, showing a stronger antitumor response compared to G2. This response was characterized by a high infiltration of CD8+ T and PD-1+ cells, and a likely prevalence of CD4+ Th1 cells as suggested by a low T CD4/CD8 ratio. This led to a marked tumor elimination represented by nearly 70% of tumor necrosis, and a 3-fold reduction of both tumor volume and mitotic index. Interestingly, anti-PD1 seemed to successfully enhance an important part of the vaccine’s antitumor response, indicated by 50% less TAMs infiltration and 10% more necrosis, though not significantly improving the overall antitumor effect of the vaccine. These limitations of the combined therapy may be overcome by using a higher anti-PD-1 dose and administering the two treatments at different times. Furthermore, the positive and promising effects of applying both treatments individually and combined, plus the vaccine’s low-cost and simple preparation method, encourage us to continue this study. The vaccine should be evaluated in combination with other checkpoint inhibitors and/or other target-specific compounds that can lead to new, highly effective, less toxic, personalized breast cancer immunotherapies.

Citation Format: Maria Jose Godoy Calderon, Eglys González Marcano, Ana Federica Convit. Combined therapy with an autologous tumor cells/bacillus Calmette-Guérin/formalin-based vaccine plus anti-PD-1 inhibitor enhances the antitumor response in a 4T1 breast cancer model [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B112.
Abstract B127: NT219, a novel dual inhibitor of STAT3 and IRS1/2, converts immuno-oncology resistant tumors to responders
Authors: Reuveni Hadas , Haviv Izhak , Kupershmidt Lana
Published: February 2019

Feedback activation of STAT3 and IGF1R/IRS plays a prominent role in mediating drug resistance to a broad spectrum of targeted cancer therapies and chemotherapies. Both the IRS1/2 and STAT3 are major signaling junctions regulated by various oncogenes, and altered during EMT and drug resistance. STAT3 has also been known to play an active role in immune-evasion of tumors and inhibition of STAT3, both in the tumor, as well as in the tumor’s microenvironment, may therefore potentiate immune attack on the tumor. NT219 is a dual inhibitor of STAT3 and IRS1/2 developed by TyrNovo Ltd. to overcome cancer drug resistance. NT219 inhibits STAT3 phosphorylation and eliminates IRS1/2 in a unique 3-step mechanism: dissociation of IRS1/2 from the IGF1 receptor, induction of IRS1/2 serine phosphorylation, and subsequent degradation by the proteasome. We recently demonstrated that the inhibition of both IRS and STAT3 are required and essential for overcoming drug resistance. NT219 efficacy was demonstrated in patient-derived tumor xenograft (PDX) models of multiple cancer types: melanoma, sarcoma, pancreatic, colon, lung, and head and neck when added on to the approved therapies. In these models, NT219 overcame acquired resistance to several oncology drug families: inhibitors of EGFR (Tarceva®, Erbitux®, Tagrisso®), MEK (Mekinist®), mutated-BRAF (Zelboraf®), mTOR (Afinitor®) as well as with chemotherapy agents (Gemzar®, 5FU, oxaliplatin). We recently demonstrated that NT219 works in synergy also with immune-oncology therapies. By using double autologous PDX models we demonstrated that NT219 converted non-responding tumors to responders to Keytruda®. It also enhanced the immunotherapeutic potential of cetuximab. FACS analysis showed increased tumor infiltrated lymphocyte population following treatment with NT219 and the combination of NT219 and the immuno-oncology drug.The unique mode of action of NT219 may open a new avenue of combined targeted therapies in a wide range of malignancies, and has the potential to expand response duration and target patient population to the applicable drugs.

Citation Format: Hadas Reuveni, Izhak Haviv, Lana Kupershmidt. NT219, a novel dual inhibitor of STAT3 and IRS1/2, converts immuno-oncology resistant tumors to responders [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B127.
Abstract B130: Induced lymphangiogenesis enhances antigen-specific T-cell response in anticancer vaccination
Authors: Sasso Maria Stella , Hauert Sylvie , Briquez Priscilla , Wang Yue , Ishihara Jun , Hubbell Jeffrey A. , Swartz Melody A.
Published: February 2019

Tumor-associated lymphatic vessels play multiple and complex roles in the regulation of antitumor immunity. In both mouse and human melanomas, lymphatic markers correlate with increased tumor immune infiltrates and enhanced responsiveness to immunotherapy. Despite the immune-promoting functions of lymphatics in cancer immunotherapy, manipulation of lymphatic vessels directly within the tumor site appears controversial as a therapeutic strategy, due to the correlation of tumor lymphatic growth (lymphangiogenesis) with cancer cell dissemination and metastasis. Here, we sought to manipulate and exploit lymphatic vessels remotely from the tumor by developing and characterizing a lymphangiogenic cancer vaccine. Lymphangiogenic whole-cell vaccines were formulated using lethally irradiated tumor cells overexpressing the pro-lymphangiogenic growth factor VEGF-C in combination with topically retained immune adjuvants (VEGFC vax). In the B16F10 and B16F10-OVA mouse melanoma models, VEGFC vax induced extensive lymphatic growth at the cutaneous injection site, as assessed by both flow cytometry and histologic analysis. Induced lymphangiogenesis increased the rate of fluorescent microbead transport from the skin to the draining lymph nodes, suggesting an enhanced antigen trafficking. In addition, VEGF-C overexpression drove the recruitment in the vaccine injection site of naïve T-cells (responsive to lymphatic-derived CCL21), which could be primed in situ. Compared to a control VEGF-C-free vaccine formulation, VEGFC vax induced a broader T-cell response targeting multiple mouse melanoma antigens (including tumor neoantigens), as assessed by IFNγ ELISPOT following antigen-specific stimulation. In a prophylactic setting, VEGFC vax provided complete protection against B16F10 melanoma challenge, outperforming both the control vaccine and a GM-CSF-secreting whole-cell vaccine (GVAX). Overall this study provides a proof of concept for the use of VEGF-C as an immunostimulatory agent in cancer vaccines, based on its ability to activate and expand lymphatic vessels and consequently promote local immune cell trafficking, antigen transport and T-cell activation. In addition, we here developed and characterized a whole-cell VEGF-C vaccine formulation with potential for clinical translation.

Citation Format: Maria Stella Sasso, Sylvie Hauert, Priscilla Briquez, Yue Wang, Jun Ishihara, Jeffrey A. Hubbell, Melody A. Swartz. Induced lymphangiogenesis enhances antigen-specific T-cell response in anticancer vaccination [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B130.
Abstract B135: Novel immunotherapeutic strategy based on the immunopathologic properties of cancer stem cells
Authors: Torigoe Toshihiko , Hirohashi Yoshihiko
Published: February 2019

Cancer stem-like cells (CSCs) are small subpopulation of cancer cells that contribute to cancer initiation and progression. These cells have a huge impact in the field of cancer therapy since they are resistant to standard chemoradiotherapy and responsible for disease recurrence. We have studied immunopathologic properties of CSCs derived from colon, breast, and gynecologic cancers to develop a novel CSC-targeted immunotherapy. #1 CSC-specific antigens: CSCs express several germ cell-specific genes. Remarkably, some of them were involved in tumor initiation capacity of CSCs and highly immunogenic since specific cytotoxic T-cells (CTLs) were efficiently induced from peripheral blood lymphocytes. The CTLs exerted specific cytotoxicity against CSCs. Adoptive transfer of the CSC-specific CTLs efficiently protected tumor growth in mouse models. #2 Fibroblastic niche of CSCs: Cancer-associated fibroblasts (CAFs) have important roles in tumor microenvironment. CSCs recruited fibroblasts, which enhanced tumor initiation capacity and immune resistance of CSCs. Gene expression analysis revealed that fibroblast growth factor 4 (FGF4) and FGFR2, a receptor for FGF4 were involved in the fibroblastic niche of CSCs. #3 Innate PD-L1 expression of CSCs: Some CSCs constitutively expressed PD-L1 on the cell surface through activation of PI3K/AKT signaling. Based on these unique properties of CSCs, we propose immunotherapeutic and immunoprophylactic strategy targeting CSCs.

Citation Format: Toshihiko Torigoe, Yoshihiko Hirohashi. Novel immunotherapeutic strategy based on the immunopathologic properties of cancer stem cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B135.
Abstract B139: Plant viral particle vaccine induces a potent antitumor response through induction of antigen-specific T-cells and overcoming an immunosuppressive tumor microenvironment
Authors: Wang Chuan , Wang Yidao , Allen Alex J. , Jobsri Jantipa , Thomas Gareth J. , Ottensmeier Christian H. , Savelyeva Natalia
Published: February 2019

In order to develop an effective vaccination strategy for cancer, it is necessary to induce robust T-cell responses and to overcome the immunosuppressive tumor microenvironment (TME). To induce anticancer immunity, we focused on a plant viral particle (PVP) that contains-single strand RNA (ssRNA) as a flexible and economical platform to deliver cancer antigens. Initially using PVP loaded with SIINFEKL, we demonstrated the induction of a rapid CD8 T-cell response leading to a significant therapeutic effect in the B16 tumor model. The induction of high levels of CD8 T-cells was also achieved when clinically relevant cancer antigens were used with lysis of human cancer cells expressing these antigens. Further, PVP loaded with whole protein antigen induced CD8 and CD4 T-cell responses against the epitopes delivered through the whole antigen and these cleared established tumors. The mechanisms behind the high immunogenicity of the PVP vaccine revealed that this ssRNA containing vaccine activated the immune mechanisms closely resembling the natural antiviral defence. In a murine model, the immune induction was through the activation of TLR7, leading to release of type I IFN by plasmacytoid dendritic cells and activation of conventional DCs (cDCs) followed with the priming of Th1 immunity. These findings were further supported by experiments using human monocytes derived DCs (moDCs) stimulated in vitro with PVP. The stimulated moDCs released Th1-polarizing cytokines and chemokines, and upregulated CD40 and B7 family surface molecules. Further investigation of the TME demonstrated that cDCs, MHC-II high tumor associated macrophages (TAMs) and tumor-infiltrating neutrophils were recruited into the tumor bed 24 hours after vaccination. While MHC-II low TAMs and myeloid-derived suppressor cells were down-regulated in response to vaccination. This was followed by the increase of tumor-infiltrating lymphocytes (TILs) specific for endogenous tumor antigens. In conclusion, the PVP vaccine is an effective platform to induce T-cells specific to the delivered cancer antigens, and furthermore benefit from its ability to convert an immunosuppressive TME towards an immunostimulatory environment, facilitating the endogenous T-cell epitopes spread.

Citation Format: Chuan Wang, Yidao Wang, Alex J. Allen, Jantipa Jobsri, Gareth J. Thomas, Christian H. Ottensmeier, Natalia Savelyeva. Plant viral particle vaccine induces a potent antitumor response through induction of antigen-specific T-cells and overcoming an immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B139.
Abstract B142: Activation of endogenous anergic self-specific CD8+ T-cells by polymeric nanoparticles for enhanced cancer immunotherapy
Authors: Yin Qian
Published: February 2019

While the human immune system is often able to protect the body from infectious pathogens, it has multiple mechanisms to inhibit mounting an immune system against what it perceives as “self” and thus often fails to eliminate cancer cells since they seem to have the characteristics of “self.” Our lab found that cytotoxic CD8+ T-cells that recognize self antigens in the peripheral blood of healthy humans were present in frequencies similar to those specific for non-self antigens, but these self-specific CD8+ T-cells are resistant to activation and/or expansion. We hypothesized that tumor-infiltrating CD8+ T-cells recognizing self antigens are often in an anergized state but they could be activated with the appropriate immunostimulatory signals to augment antitumor immunity. Thus, we developed a poly(lactide-co-glycolide) nanoparticle (PLGA NP) based immunostimulatory platform that is surface-functionalized with monoclonal anti-CD28 antibody (Ab), and encapsulated interleukin-2 (IL-2), toll-like receptor-2 (TLR2) agonist and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in the hydrophobic core. This platform enables the encapsulated immunostimulatory drugs to be continuously released in the tumor microenvironment to activate the tumor infiltrating self-specific CD8+ T-cells in order to enhance antitumor immune response while minimizing the rapid diffusion of these drugs into other parts of the body, greatly reducing the systemic toxicities. Our results demonstrated that the mice bearing melanoma or colon cancers treated with the stimulatory PLGA NP have elicited potent T-cell response, resulting in marked tumor regression and increased overall survival. The developed PLGA NPs are also capable of generating an immunologic memory that prevented tumor growth after rechallenging the survivor mice with the same cancer cell type. Moreover, the developed PLGA NPs can be combined with checkpoint blocking antibodies to achieve the synergistic effect, significantly prolonging the overall survival. We also applied the single-cell TCR sequencing technique to assess both T-cell receptor and phenotypic characteristics of tumor-infiltrating CD8+ T-cells after NP stimulation. The result was further combined with yeast-display libraries of peptide-MHC to identify tumor-specific antigens.

Citation Format: Qian Yin. Activation of endogenous anergic self-specific CD8+ T-cells by polymeric nanoparticles for enhanced cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B142.
Abstract B145: Identification of tumor cell intrinsic immune evasion mechanisms
Authors: Reviriego Carmen Ballesteros , Speak Anneliese O. , Turner Gemma , Iyer Vivek , Parts Leopold , Adams David J.
Published: February 2019

The ability of cells of the immune system, in particular CD8+ cytotoxic T-cells and natural killer (NK) cells, to kill tumor cells in vivo is now being exploited in the clinic with the advent of immunotherapy. While these therapies are effective in a subset of patients the emergence of tumor resistance is now being observed in the clinic. Several studies have investigated tumour evasion from CD8+ cytotoxic T-cell directed killing; however, it is not known if the same mechanisms apply for the tumor cells to evade NK cells. For instance, one major pathway of CD8+ cytotoxic T-cell resistance is via alteration of the MHC class I antigen presentation pathway which could in theory allow the tumor cells to become more sensitive to NK cell mediated killing due to “loss of self.” We have investigated tumor cell intrinsic NK cell resistance and sensitivity pathways using genome-wide CRISPR-Cas9 loss of function in vitro screens. These screens have confirmed the essential role of cell adhesion via ICAM1 in enabling tumor cell killing by NK cells. We have also identified other novel pathways that could be manipulated to enhance tumor cell cytotoxicity and the development of alternative and/or additive therapeutic strategies in the clinic.

Citation Format: Carmen Ballesteros Reviriego, Anneliese O. Speak, Gemma Turner, Vivek Iyer, Leopold Parts, David J. Adams. Identification of tumor cell intrinsic immune evasion mechanisms [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B145.
Abstract B167: Antigen-presenting cells as coordinators of T-cell responses to gut microbiota
Authors: Kedmi Ranit , Mesa Kai , Littman Dan
Published: February 2019

The immune system in the gut mucosa must balance tolerance to harmless diet and microbial antigens with the ability to mount effective inflammatory responses to diverse invasive pathogens. These requirements are reflected in the functions of distinct subsets of T-cells whose differentiation is guided by diverse cytokines secreted by antigen-presenting cells (APCs). In recent years, different APCs subsets have been proposed to guide CD4+ T-cell polarization and to induce primary T-cell responses in lymph nodes versus secondary responses in tissues. However, the exact roles of APC subsets in mediating T-cell responses to gut microbiota are still under debate and have not been thoroughly addressed. We previously showed that the commensal microbe segmented filamentous bacteria (SFB) induces antigen-specific Th17 cells, while Helicobacter hepaticus (Hh) induces an antigen-specific Treg response. Using those microbial models, I am now studying the contribution of APC subsets to specific T-cell response. I utilize several different approaches to track APC subsets that presents the relevant microbial peptides in the mesenteric lymph node and to characterize their contribution to T-cell priming and polarization. A better understanding of which APCs participate in the differentiation of each category of T-cell will enable in the future to develop better tools to educate T-cells to fight cancer.

Citation Format: Ranit Kedmi, Kai Mesa, Dan Littman. Antigen-presenting cells as coordinators of T-cell responses to gut microbiota [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B167.
Abstract B169: Engineering phase separation of multivalent signaling proteins through the solubility of folded protein domains
Authors: Kim Jonggul , Ditlev Jonathon , Rosen Michael K.
Published: February 2019

Similar to three-dimensional membraneless organelles, it has been demonstrated that T-cell LAT signaling clusters form through multi-valency driven phase separation. This phase separated cluster was found to promote Arp2/3-complex driven actin assembly in vitro and correlated with increased ERK phosphorylation in cells. However, in this initial study, perturbations used to study phase separation also affect interactions known to be important for T-cell signaling, convoluting the impact of phase separation on signaling with effects due to changes in canonical biochemical interactions. To understand more precisely the role of phase separation in T-cell signaling, I will alter the solubility of folded domains through mutations of surface charged residues to modulate their phase separation propensities, independent of their high-affinity ligand binding interactions. To demonstrate the validity of this approach, I have first applied it to a synthetic system, based on multivalent interactions between the small ubiquitin-like modifier (SUMO) and the SUMO interaction motif (SIM), which will enable us to provide design principles that can be used on T-cell receptor signaling proteins. I demonstrate that it is possible to specifically alter the solubility of folded domains without altering their canonical molecular interactions. The solubility of individual folded domains serves as a predictor of the concentration threshold of phase separation, and phase separation can either be enhanced or abolished with a single mutation. Principles gained from this work on SUMO/SIM will be applied to multivalent proteins involved T-cell receptor signaling, namely Grb2 and Nck1, and the relationship between phase separation and signaling will be firmly established with future implications for rational engineering of T-cells.

Citation Format: Jonggul Kim, Jonathon Ditlev, Michael K. Rosen. Engineering phase separation of multivalent signaling proteins through the solubility of folded protein domains [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B169.
Abstract B173: G protein coupling signaling as regulators of dendritic cell maintenance and function in immune responses
Authors: Liu Dan , Cyster Jason G.
Published: February 2019

Dendritic cells (DCs) are well known as the professional antigen presenT-cells (APCs), which can scan peripheral tissues where they can recognize, uptake, process and present all kinds of antigens, including pathogens and tumor antigens, to antigen specific naïve T-cells within the lymphoid organs. In these processes, DCs form a remarkable bridge between innate and adaptive immunity by interacting with various lymphoid and myeloid cells. DCs originate from the bone marrow hematopoietic progenitor cells and DC precursors migrate from blood to tissues for developing into immature DCs. Upon activation, DCs migrate to stimulate T-cells and induce immune responses to protect our bodies. However, the mechanism for maintenance and activation of DCs on the local microenvironment is largely unknown. As GPCR signaling has important roles in many cellular process, including cell division, survival, migration and adhesion. Using competitive mixed BM chimaera, I checked some molecules involved in GPCR signal pathways, and found that geneA was required intrinsically in the maintenance of CD4+ conventional DCs (cDCs) in the spleen, as evidenced by observation that geneA-deficient CD4+ DCs were dramatically decreased in spleen but increased in blood. By in vivo 3min CD45-PE labeling assay, cells exposed to the vascular compartment in spleen can be selectively labeled. With this method, I found without geneA, CD4+ DCs had disadvantage to be maintained in the blood-exposed region within the spleens under the shear flow stress. I further showed that geneA-deficient CD4+ DCs had disadvantage to uptake blood-derived Sheep-blood cells (SRBCs). Besides this stimulation, this signal also controlled CD4+ DCs activation in response to different kinds of TLR stimulators. By OT-II adoptive transfer system, geneA-expressing DCs were required to support T-cell proliferation and differentiation efficiently. Therefore, these results demonstrate a key role of G protein-coupled receptor signaling in promoting the maintenance and activation of DCs, and reveal a mechanism in DC positioning in vivo. Thus, it will broaden our understanding of GPCR signaling in DC immunology, which is pivotal for modulating DCs for vaccines and therapies against pathogens, autoimmune diseases and tumors.

Citation Format: Dan Liu, Jason G. Cyster. G protein coupling signaling as regulators of dendritic cell maintenance and function in immune responses [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B173.
Abstract B174: Co-expression of stimulators and inhibitors of T-cell activation in melanoma
Authors: Maniyar Rachana , Freund Robert , Malhotra Aryan , Chakraborty Sanjukta , Geliebter Jan , Wallack Marc , Tiwari Raj K.
Published: February 2019

Melanoma, one of the most aggressive skin cancers, has steadily been on the rise over the last three decades. With limited treatment options, the recent impetus in immuno-oncology drugs, especially checkpoint inhibitors, has changed the treatment landscape. While anti-CTLA4 and anti-PD1 demonstrated success in clinic, the search for additional targets is needed to enable potent comprehensive curative combination therapies. Our laboratory has characterized and screened five primary patient-derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM, and GLM-2. While immunomodulatory molecules are canonically present on antigen presenting cells and T-cells, increasing evidence suggests that they are not expressed in isolation. In an effort to identify immunomodulatory molecules expressed on our primary melanoma cells, a comprehensive RT-PCR screen of 29 co-inhibitory and co-stimulatory molecules was carried out. Several molecules including CD160, CD226, TIM1, HVEM, and BTLA were seen to be differentially expressed in melanoma cells compared to normal melanocytes at the mRNA level. Western blots and immunocytochemistry validated the differential expression of these molecules at the protein level. 50-80% of melanoma cases are positive for the BRAFV600E mutation, and are treated with a small molecule inhibitor of the mutated BRAF, vemurafenib (PLX4032). Treatment of these cells with PLX4032 led to an upregulation of transcription factors MITF and AP-1, as well as immunomodulatory molecules, CD160, CD226, TIM1, HVEM, and BTLA, a phenomenon seen only in cells positive for the BRAFV600E lesion. MITF and AP-1, owing to the binding sites present in the promoter regions of these molecules, can drive their expression upon treatment with PLX4032. These additional immune-regulatory molecules of T-cell activation and/or immune tolerance mechanisms are potential targets for a combination therapy with PLX4032 in melanoma patients positive for the BRAFV600E genetic lesion. Our future directions aim to elucidate the role of these molecules on the tumor cells and devise an effective combination with small-molecule inhibitors and immunotherapies.

Citation Format: Rachana Maniyar, Robert Freund, Aryan Malhotra, Sanjukta Chakraborty, Jan Geliebter, Marc Wallack, Raj K. Tiwari. Co-expression of stimulators and inhibitors of T-cell activation in melanoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B174.
Abstract B177: Analysis of immunogenic cell death (ICD) induced in multiple myeloma cells
Authors: Matsushita Maiko , Kashiwazaki Sho , Kamiko Satoshi , Uozaki Ryo , Ichikawa Daiju , Hattori Yutaka
Published: February 2019

Background: Development of novel drugs, such as immunomodulatory drugs (IMiDs), proteasome inhibitors (PIs), or monoclonal antibodies, has prolonged survival of multiple myeloma (MM) patients. However, high-risk patients harboring del 17, t(14;17), or t(11;14) still have poor prognosis. Therefore, clarification of the drugs that could potentiate the effect of these treatments is necessary. Several drugs have been reported to cause cell surface expression of calreticulin (CRT), release of high mobility group box 1 (HMGB 1) and ATP, activating an immune response, which is called immunogenic cell death (ICD). However, there is almost no information on ICD in MM. The purpose of this study is to investigate whether anti-MM drugs can induce not only direct cell-killing effect but also immunomodulatory effects in high-risk MM cells. Methods: High-risk myeloma cell line, MUM24 was treated with dexamethasone, melphalan, lenalidomide, bortezomib, carfilzomib, and panobinostat. Expression of cell surface CRT and HMGB1 release were detected using flow cytometry and Western blotting, respectively. We then co-cultured DiD-labeled monocyte-derived dendritic cells with GFP-induced MUM24 treated with each drug and evaluated phagocytosis of DCs using fluorescence microscope. Results: After treatment with drugs, bortezomib and carfilzomib induced higher expression of CRT and release of HMGB1 in MUM24 compared with other drugs. We also observed that DCs could engulf MUM24 cells treated with carfilzomib. Conclusion: Our results suggest that PIs, such as bortezomib or carfilzomib, could not only kill MM cells but also induce ICD. These effects are expected to improve the prognosis of MM patients by evoking strong immune response against MM cells.

Citation Format: Maiko Matsushita, Sho Kashiwazaki, Satoshi Kamiko, Ryo Uozaki, Daiju Ichikawa, Yutaka Hattori. Analysis of immunogenic cell death (ICD) induced in multiple myeloma cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B177.
Abstract B188: CMTM6, beyond a regulator of PD-L1 expression
Authors: Sun Chong , Mezzadra Riccardo , Gomez-Eerland Raquel , Hofland Ingrid , Peters Dennis , Broeks Annegien , Horlings Hugo M. , Wu Wei , Heck Albert J. R. , Schumacher Ton N.M.
Published: February 2019

Expression of PD-L1 by tumor cells and by infiltrating immune cells represents a critical feature of cancer immune evasion. In line with this, clinical targeting of the PD-1 – PD-L1 axis forms one of the most significant breakthroughs in cancer treatment of the past years. To study the mechanisms that regulate PD-L1 expression, we have previously performed a FACS-based genetic screen and identified CMTM6, a functionally-uncharacterized protein, as a novel regulator of PD-L1 protein levels. Furthermore, we observed that CMTM6 forms a molecular complex with PD-L1 and enhances the ability of PD-L1-expressing tumor cells to inhibit T-cells. However, apart from this important role for CMTM6 in PD-L1 regulation, our understanding of this protein remains highly limited. To further characterize CMTM6, we surveyed its expression in major tissues and organs in the human body. Analysis by IHC and flow cytometry shows thaT-cells in immune-privileged tissues and in primary and secondary lymphoid organs express CMTM6. In addition, we explored other biologic roles of CMTM6. We found CMTM6 positively regulates and co-immunoprecipitates with the co-stimulatory molecule CD58. Taken together, these findings indicate a broader role for CMTM6 in immune modulation.

Citation Format: Chong Sun*, Riccardo Mezzadra*, Raquel Gomez-Eerland*, Ingrid Hofland, Dennis Peters, Annegien Broeks, Hugo M. Horlings, Wei Wu, Albert J. R. Heck, Ton N.M. Schumacher. CMTM6, beyond a regulator of PD-L1 expression [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B188.
Abstract B196: Examining proteasome sensitivity of lymphoid malignancy: Ubiquilin 1 (Ubqln1) is required for BCR-induced proliferation of B cells
Authors: Whiteley Alexandra M. , Brown Eric , Finley Daniel
Published: February 2019

Careful regulation of protein levels is essential to cellular life, and the ubiquitin proteasome system is a major way in which cells coordinate the degradation of proteins. In addition to regulating steady state protein degradation, the ubiquitin proteasome system assists in major proteome reorganizations, including the response to cellular stresses and the completion of major milestones such as differentiation, stimulation, and cell division. Lymphocytes are a useful model with which to understand the regulation of protein degradation because they undergo dramatic proteome reorganization upon receptor:ligand interactions, and because some lymphocytic malignancies are uniquely sensitive to treatment with proteasome inhibitors. We have previously shown that B lymphocytes require Ubiquilin 1 (Ubqln1), an extrinsic proteasome receptor, for their proliferation downstream of BCR stimulation. Ubqlns help to facilitate the degradation of ubiquitinated protein by simultaneously binding the cap of the proteasome and ubiquitinated client proteins, but until recently the clients of Ubqln1, as well as the consequences of Ubqln1 loss, were unknown. The lack of Ubqln1 resulted in accumulation of mislocalized mitochondrial proteins and induced a block in protein synthesis downstream of BCR stimulation, which resulted in cell cycle inhibition. This work implicated a role for Ubqln1 both in immune activation as well as B cell malignancy. Unexpectedly, TLR stimulation was normal in Ubqln1-deficienT-cells. Two major hypotheses for this specificity involve unique signal transduction pathway activation, and overexpression of mitochondrial proteins downstream of BCR activation. In fact, dysregulation of BCR signal transduction cascades and upregulation of OXPHOS genes define common and partially overlapping subtypes of diffuse large B-cell lymphoma (DLBCL). Currently, we aim to resolve these hypotheses with both in vivo and in vitro approaches. These studies will sharpen our understanding of B-cell cancer susceptibility to perturbation of the ubiquitin proteasome system and of Ubqln1 function.

Citation Format: Alexandra M. Whiteley, Eric Brown, Daniel Finley. Examining proteasome sensitivity of lymphoid malignancy: Ubiquilin 1 (Ubqln1) is required for BCR-induced proliferation of B cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B196.
Abstract B201: Exploration and exploitation of macrophage death pathways: Infection by Mycobacterium tuberculosis as a model
Authors: Zander Ryan , Schauder David , Xin Gang , Nguyen Christine , Wu Xiaopeng , Cui Weiguo
Published: February 2019

Macrophages can impact the development of tumors and their responses to therapy. However, we lack a full understanding of how macrophage activation and survival are regulated and might be therapeutically modulated. To address these questions, we turned to a host-pathogen interaction that is likely to have impacted the evolution of these processes: infection by Mycobacterium tuberculosis (Mtb), the leading cause of death from infection. Once internalized by macrophages, Mtb resists killing by macrophages and replicates within them. Ultimately, Mtb infection results in death of macrophages, allowing dissemination of the pathogen to other cells. To explore how Mtb induces macrophage cell death and how macrophage cell death might impact host defense against Mtb, we performed a genome-wide CRISPR-Cas9 recessive genetic screen in RAW264.7 macrophages. We discovered that the absence of components of the type I interferon signaling pathway, including the IFN-α/β receptor (IFNAR), delays Mtb-induced cell death. It is known that Mtb infection induces macrophages to produce type I interferon. Our finding directly links type I interferon signaling pathway and Mtb-induced macrophage death. We are currently working on the mechanism of the type I IFN-induced death of bacterially-stimulated macrophages. Meanwhile, we are testing blockers of this pathway as host-directed therapy against TB and have seen striking protective effects of anti-IFNAR1 mAb in Mtb-infected mice, whether the mAb is administered before or after infection. Further exploration of the protective effect of anti-IFNAR mAb could point to an antibody-based, host-directed therapy. Acknowledgment: We thank Prof. R. Schreiber, Washington University, for facilitating access to anti-IFNAR mAb.

Citation Format: Ryan Zander, David Schauder, Gang Xin, Christine Nguyen, Xiaopeng Wu, Weiguo Cui. Exploration and exploitation of macrophage death pathways: Infection by Mycobacterium tuberculosis as a model [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B201.
Abstract B202: Defining the role of IL-27 signaling on responses to metastatic melanoma
Authors: Phan Anthony T. , Hunter Christopher A.
Published: February 2019

Checkpoint blockade and cellular therapies for cancer treatment have yielded impressive clinical results. These approaches harness the ability of T-cells, critical mediators of the body’s immune response to infection, to eliminate tumors. Entry of T-cells into the tumor microenvironment exposes them to an array of regulatory signals that impact T-cell differentiation and function. The immunomodulatory cytokine, interleukin 27 (IL-27), produced by tumor cells and immune cells, is a critical regulator of the immune system. A complex literature exists regarding the role of IL-27 in tumor responses and the conflicting conclusions from these studies can be attributed to differences in tumor models and a lack of lineage-specific genetic tools to dissect the impact of IL-27 signaling on particular cell types. We find that IL-27 receptor alpha (WSX1)-deficient mice exhibit reduced tumor burdens following intravenous administration of B16 melanoma in comparison to wildtype mice. Further, the reduced number of macroscopic nodules corresponds with reduced tumor area within the lungs. Analysis of IL-27 reporter mice reveals a population of nonalveolar macrophage/monocytes to be the primary source of IL-27 in the lung as early as day 10 following B16 melanoma challenge. The production of IL-27 in the lung following tumor challenge and the improved resistance to intravenous dissemination of B16 melanoma by WSX1-deficient mice suggests production of IL-27 suppresses immune responses to melanoma within the lung. In line with these data, we find that tumor nodules in the lungs of WSX1-deficient mice have increased infiltration of CD3+ cells observed by histologic analysis in comparison to tumor nodules in wild-type mice. These data support a model where IL-27 suppresses the infiltration or activity of T-cells responding to metastatic melanoma and suggest that novel strategies targeting IL-27 signaling in the tumor microenvironment may synergize with current immunotherapy approaches to improve T-cell responses to tumors.

Citation Format: Anthony T. Phan, Christopher A. Hunter. Defining the role of IL-27 signaling on responses to metastatic melanoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B202.
Abstract IA12: CAR T-cell therapy for lymphoma and multiple myeloma
Authors: Kochenderfer James N.
Published: February 2019

Chimeric antigen receptors (CARs) are fusion proteins that incorporate antigen-binding domains and T-cell signaling domains. The first CAR T cells to be shown to be clinically effective targeted the B-cell antigen CD19. Anti-CD19 CAR T cells have been effective against all major types of B-cell lymphomas, and anti-CD19 CAR T cells have now obtained Food and Drug Administration (FDA) approval for treatment of the most common type of lymphoma, diffuse large B-cell lymphoma. This approval was based on clinical results that showed an approximately 50% complete remission rate of anti-CD19 CAR T-cell therapy for diffuse large B-cell lymphoma. These responses have been durable. Eleven of twelve patients who obtained complete remissions on a phase 1 trial of anti-CD19 CAR T cells are still progression-free 13 to 36 months after treatment (Kochenderfer et al. Journal of Clinical Oncology, 2017). Even more durable remissions have occurred in a small number of patients in an early cohort of anti-CD19 CAR therapy. Four patients with remissions of 38 to 56 months will be presented. These patients are particularly interesting because 3 of the 4 patients with long remissions have had recovery of normal B cells while the complete remissions have continued. This result shows that remissions of diffuse large B-cell lymphoma can continue after disappearance of a functional anti-CD19 CAR T-cell response. Beyond CD19, active CAR T-cell therapy for multiple myeloma has been developed by targeting B-cell maturation antigen (BCMA). Anti-BCMA CAR T cells have undergone rapid clinical development since first being reported in 2013 (Carpenter et al. Clinical Cancer Research, 2013). Clinical results of the first-in-humans clinical trial of anti-BCMA CARs (Brudno et al. Journal of Clinical Oncology, 2018) will be presented, and future plans for multiple myeloma CAR T-cell therapies will be discussed.

Citation Format: James N. Kochenderfer. CAR T-cell therapy for lymphoma and multiple myeloma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA12.
Abstract IA18: Inactivation of DNA repair to improve immune surveillance
Authors: Bardelli Alberto
Published: February 2019

When metastatic cancers are challenged with targeted agents almost invariably a subset of cells insensitive to the drug emerges. As a result, in most instances, targeted therapies are only transiently effective in patients. Strategies to prevent or overcome resistance are therefore essential to design the next generation of clinical trials. How can we overcome the near-certainty of disease recurrence following treatment with targeted agents? Addressing this question means considering as a target not “only” individual oncogenes but also the evolving nature of human tumors. We used colorectal cancer (CRC) as a model system to test the hypothesis that by understanding tumor’s evolution, the emergence of drug resistance can be controlled. We find that to have long-term efficacy, the use of targeted therapies must take into account the continuous evolution of cancer cells, that is to say, therapies must adapt to tumor evolution. Another approach is to unleash the ability of the immune system to recognize drug resistant cells. We tested this possibility in syngeneic mouse models of CRC sensitive to targeted therapies. Our findings indicate that inactivation of DNA repair mechanisms and manipulation of mutational loads can trigger immune surveillance and prolonged therapeutic responses. We postulate that rationally combined targeted and immuno-therapies can restrain tumor evolution, and can limit the emergence of drug resistance thus leading to long-term responses.

Citation Format: Alberto Bardelli. Inactivation of DNA repair to improve immune surveillance [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA18.