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{"title":"Abstract B167: Antigen-presenting cells as coordinators of T-cell responses to gut microbiota","authors":"Ranit Kedmi, Kailin R. Mesa, D. Littman","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B167","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B167","url":null,"abstract":"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.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"18 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"114169300","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B166: Mass spectrometric characterization of MHC peptides on therapeutic EBV transformed primary lymphoblastoid cells from HLA A*11 patients","authors":"Amit Jain","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B166","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B166","url":null,"abstract":"EBV transformed lymphoblastoid cell lines (LCL) derived from patients with Epstein Barr virus (EBV)-related cancer have been used as \"feeder cells\" after terminally irradiation in multiple cell therapy protocols targeting EBV-related cancers. These cells are subsequently co-cultured with patient peripheral blood mononuclear cells (PBMC) and are sufficient expansion of autologous cell product, given therapeutically back to patients. LCL are known to present both host genome derived cancer-associated antigens relevant for cancer, alongside EBV antigens. PBMC that are co-cultured with LCL have been used in multiple small studies across multiple EBV related cancers. This study was performed in the context of LCL used as feeder cells in the context of EBV-related nasopharyngeal cancer that was treated with autologous cell product after co-culture with LCL as part of a phase II clinical trial in Singapore. The aims of this study were, firstly, to perform unbiased identification of MHC peptides using mass spectrometry; secondly, to map the peptides to respective HLA alleles using publicly available HLA peptide binding prediction algorithms; and thirdly, to look specifically at HLA A*11 allele specific EBV-derived peptides. 6 patients with HLA A*11 were identified and LCL from these patients were grown in cell culture for 1-3 months each, until 5E8 cells were available. Cells were treated in suspension with acidification in order to enable acid elution of MHC peptides. Mass spectrometric analysis was performed for unbiased evaluation of MHC peptides. Based on the consensus peptide sequences derived from 8-mer and 9-mer peptides, HLA A*11:01 appeared to be a dominant allele with at least 20% of peptides having basic end residues that are typical for binding to HLA allele. Between 3,000 to 5,000 distinct peptide sequences across lengths characteristic for both class I and II MHC alleles were identified for each sample, including multiple peptides derived host genome DNA, and some from EBV proteins including LMP1, BARF1, EBNA6, and TTK. There was some overlap of peptides seen across the samples. For example, 2 patient samples, with 3,636 and 4,033 distinct peptides identified respectively, had 1,025 overlapping peptides. 2 patient samples with HLA A*11:01 and HLA A*11:02 respectively, had 1 overlapping EBV protein derived set of peptides that demonstrated differences in trimming of peptides derived from the same epitope across these 2 HLA classes. Only 1 EBV protein was found to be consistently represented in the MHC peptidome across the patient samples, with the same epitope represented across all the samples within the scope of this study. This small study demonstrates that mass spectrometric analysis of large numbers of cells is able to directly deconvolute the MHC peptide, and this is relevant in the context of adoptive cell therapies that utilize \"feeder cells\" that are required to reliably present clinically relevant antigens to generate autologous ce","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"99 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"116105431","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B186: Tumor cell-derived proteases contribute to antigen processing and enhance cross-presentation","authors":"N. Shields, K. Young, Christopher Jackson, S. Young","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B186","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B186","url":null,"abstract":"Tumor lysates (TLs) offer a source of both undefined tumor antigens and endogenous adjuvants, called danger-associated molecular patterns (DAMPs), to induce antitumor immune responses. TLs can therefore serve clinical applications for immunotherapy, including vaccination and as a platform for the expansion for tumor-specific T-cells for use in adoptive cell therapy. TL-based approaches offer the advantage of inducing antitumor immunity without the need to identify specific tumor antigens, a major challenge limiting the clinical application of immunotherapies for solid tumors. Immunogenic cell death (ICD) in tumor cells has proven crucial for the generation of robust antitumor immune responses, suggesting that the induction of ICD may improve the efficacy of TL-based immunotherapies. Here, we examined the immunogenicity of TLs prepared from tumor cells exposed to increasing levels of heat (37°C, 42°C and 56°C), using the MC38-OVA/OT-I system. Molecular correlates of ICD were assessed during the course of heat-treatment and in TL preparations. Notably, lethal heat-treatment (56°C) induced primary necrosis with concomitant caspase activation, calreticulin exposure, HMGB1 release and HSP upregulation. All TLs promoted maturation of bone marrow-derived dendritic cells (BMDC), as evidenced by CD80 and CD86 upregulation, and elicited protective antitumor immunity in vaccinated mice. However, only TLs prepared from lethal heat-treated tumor cells promoted cross-presentation in BMDC to elicit antigen-specific T-cell activation ex vivo. Marked proteolytic activity was observed in lethal heat-treated tumor cells, which were capable of processing antigen and binding resultant peptides. Interestingly, inhibition of broad protease activity in lethal heat-treated TLs markedly reduced cross-presentation. Selective inhibition of calpains, but not caspases, cathepsins or the proteasome, reduced antigen processing by heat-killed tumor cells.Together, this demonstrates that tumor-derived proteases can contribute to antigen processing and enhance cross-presentation. Our data suggest that in addition to DAMP release, antigenic processing by proteases in dying tumor cells can influence the immunogenicity of cell death. Citation Format: Nicholas J. Shields, Katie A. Young, Christopher Jackson, Sarah L. Young. Tumor cell-derived proteases contribute to antigen processing and enhance cross-presentation [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 B186.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"23 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"125490668","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B145: Identification of tumor cell intrinsic immune evasion mechanisms","authors":"C. Reviriego, Anneliese O. Speak, Gemma Turner, V. Iyer, L. Parts, D. Adams","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B145","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B145","url":null,"abstract":"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.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"129 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"130729928","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B184: Modulating antigen flow to control immune tolerance","authors":"Megan K. Ruhland, Edward W. Roberts, M. Krummel","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B184","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B184","url":null,"abstract":"The immune system must maintain a delicate balance between activation and tolerance. It is required to quickly respond and eliminate a variety of threats while at the same time remain unresponsive upon encountering normal self-cells or commensal bacteria. We have created a system to track antigen trafficking throughout the immune system and using this system have identified a pathway of antigen flow from peripheral tissues and tumors to the draining lymph node (LN). By tracking antigen from steady-state tissues and tumors, we find that antigen is handled differentially in the LN depending on the inflammatory context in the periphery. During pronounced inflammation, self-antigen shows altered trafficking consistent with that of tumor antigen. Interestingly, using both gut and skin systems, commensal bacteria-derived antigen displayed the unique quality of cell type specific uptake. While each of the various migratory dendritic cell (DC) types were proficient at uptake of self and tumor antigens, only migratory CD11b+ DC and macrophages showed detectable commensal antigen uptake. Additionally, tracking of commensal bacteria-derived antigen within DC showed minimal antigen trafficking to the LN, suggesting different modes of maintaining immune tolerance are at play depending on the antigen source. We find that self-antigen can drain to the LN via migratory DC but is limited from passing to resident DC populations, which appears to prevent robust self-reactive T-cell priming. However, in the case of commensal antigen, the restriction of antigen flow is early in the pathway, thus effectively keeping the LN ignorant of antigen. Identifying the signals that restrict antigen flow in cases of immune tolerance will likely provide insight into how tumor antigen availability affects immune activation. Understanding how self, commensal and tumor antigens are handled by immune cells under various contexts will help inform the development of new targeted immunotherapies that seek to activate or inhibit immune activation in cases of cancer and autoimmunity. Citation Format: Megan K. Ruhland, Edward W. Roberts, Matthew F. Krummel. Modulating antigen flow to control immune tolerance [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 B184.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"20 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"128287104","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B160: Comparative analysis of pharmacokinetics and antitumor effect between anti-PD-1 and anti-PD-L1 in mice models","authors":"H. Hatakeyama, Taiki Kurino, Hiroyuki Suzuki, Ayu Terui, T. Uehara, Y. Arano, A. Hisaka","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B160","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B160","url":null,"abstract":"Introduction: Anti-PD-1 antibodies (αPD-1 Abs) are currently used in cancer immunotherapy that block the interaction between PD-1 and PD-L1. In addition to anti-PD-1 Abs, anti-PD-L1 (αPD-L1) Abs have also been approved to inhibit the PD-1/PD-L1 axis. However, the difference between both Abs in pharmacokinetics and anti-tumor effects have not been fully understood. In this study, we analyzed the difference between both Abs in blood concentration, biodistribution and degradation in tumor-bearing mice by using αPD-1/PD-L1 Abs labeled with radioisotopes (111In/125I) and evaluated the relationship between PK and therapeutic effects. Methods: Abs were labeled with 111In via chelate agents, and labeled with 125I through covalent bond. Tumor bearing mice were prepared by s.c. inoculation with mouse colon cancer MC38 cells or mouse breast cancer MM48 cells. The labeled Abs were intraperitoneally injected into tumor-bearing mice. Tumors and organs were harvested at several time points after the injection, and radioactivities in organs were measured by a gamma counter. The accumulation of Abs was expressed as % of injected dose/g organs. Because 111In tends to be accumulated in organs due to poor permeability and 125I was eliminated from organs rapidly due to high permeability, the ratio of 125I and 111In could reflect the degradation of Abs after cellular uptake. In pharmacologic studies, Abs were intraperitoneally injected into tumor-bearing mice at day 5, 8, and 12 after tumor-inoculation. Tumor volume was evaluated to evaluate tumor progression. Results and Discussion: It was observed that αPD-L1 Ab was largely accumulated in normal tissues, especially in the spleen and liver and degraded rapidly compared with αPD-1 Ab, resulting that the blood concentration and distribution in tumors of αPD-L1 Ab tended to be low in both tumor-bearing mice models. Moreover, αPD-L1 Ab showed lower antitumor effect due to less delivered aPD-L1 Ab to tumors than aPD-1 Ab. Collectively, the PK of αPD-1/PD-L1 Abs that target the same axis were not equivalent and the selectivity of expression of target molecules in both normal tissues and tumors should be considered to optimize their therapeutic efficacy. Citation Format: Hiroto Hatakeyama, Taiki Kurino, Hiroyuki Suzuki, Ayu Terui, Tomoya Uehara, Yasushi Arano, Akihiro Hisaka. Comparative analysis of pharmacokinetics and antitumor effect between anti-PD-1 and anti-PD-L1 in mice models [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 B160.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"92 7","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"113944057","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B148: Microvilli enable efficient T-cell antigen search and ligand detection","authors":"En Cai, Kyle Marchuk, Casey Beppler, M. Krummel","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B148","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B148","url":null,"abstract":"During antigen detection, T-cells survey the surface of antigen-presenting cells (APCs), which may display mainly nonstimulatory peptide-loaded major histocompatibility complexes (pMHCs) and only rare cognate antigen in a process involving close (nanometer-scale) membrane apposition. Thus, T-cell must solve a classic trade-off between speed and sensitivity. It has long been supposed that microvilli on T-cells act as sensory organs to enable search, but their strategy has been unknown. We used lattice light-sheet microscopy and quantum dot-enabled synaptic contact mapping microscopy to show how microvilli on the surface of T-cells search opposing cells and surfaces before and during antigen recognition. We uncovered that microvilli on T-cell surfaces dynamically survey the majority of opposing surfaces within one minute through anomalous diffusion. T-cell receptor (TCR) recognition resulted in selective stabilization of receptor-occupied protrusions, which was independent of tyrosine kinase signaling and the actin cytoskeleton. We revealed that TCRs on activated T-cells were nonuniformly distributed on cell membrane: some TCRs were concentrated on microvilli, while other TCRs formed high-density patches on flatter membrane regions. Many microvilli were TCR-occupied, but a small population of microvilli were found not occupied by TCRs. TCR high-density patches moved relative to microvilli. During T-cell-APC interaction, we observed T-cell microvilli projected deep into 3D pockets formed by veil structures on the surface of dendritic cells (DC), and DC membrane also conformed to accommodate T-cell microvilli, which increased the effective close-contact area between T-cell and APC. Such scanning pattern enabled T-cells to efficiently scan more APC surface in given time. This work defines the efficienT-cellular search process against which ligand detection takes place in T-cells. Citation Format: En Cai, Kyle Marchuk, Casey Beppler, Matthew Krummel. Microvilli enable efficient T-cell antigen search and ligand detection [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 B148.","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"4 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"114817619","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B189: Tumoral STING is required for effective anticancer immunity","authors":"Manuel A. Suter, W. Y. Zhang, Muznah Khatoo, Nikki Ya Ling Tan, Chien Tei Too, Shubhita Tripathi, P. MacAry, V. Angeli, S. Gasser","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B189","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B189","url":null,"abstract":"Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that catalyses the synthesis of cGAMP, which serves as a ligand for stimulator of interferon (IFN) genes (STING). Activation of STING results in production of type I IFNs through phosphorylation of TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3). Type I IFNs are critical participants in the innate and adaptive immune recognition of cancer cells. Deficiencies in the cGAS-STING signalling pathway have been reported in many tumors. This mitigates expression of type I IFNs and may thus contribute to non-inflamed tumor microenvironment. In particular, a non-T-cell-inflamed tumor microenvironment correlates with poor patient survival. STING agonists may contribute to antitumor activity by upregulating proinflammatory cytokines and type I IFNs and various STING agonists are now being tested in clinical trials. The role of STING in immune cells is relatively well understood; however, its role in tumor cells has not yet been described in detail. Here we show that cGAS is able to bind to DNA present in the cytosol of tumor cells and subsequently induces STING signaling leading to expression of type I IFNs. Knockout of STING in mouse prostate TRAMP-C2 tumor cells resulted in higher tumor burden and reduced infiltration of immune cells such as dendritic and CD8+ T-cells into the tumor microenvironment. Consistently, treatment with the STING agonist cGAMP elevated type I IFNs levels in TRAMP-C2 cells and led to a reduced tumor volume compared to untreated control. This suggests a pivotal role of tumoral STING in antitumor immunity. However, despite intact STING expression, most tested human cancer cell lines were not responsive to various STING agonists and consequently failed to upregulate expression of type I IFNs. In contrast, all tested tumor cell lines responded to Poly(I:C)-induced TLR3 signaling, suggesting that failure to respond to cGAMP was due to a defect upstream of TBK1. Downregulation of cGAS did not render cells responsive to cGAMP, indicating that inability to respond to cGAMP is due to deficiencies of STING to activate TBK1. In the human and mouse prostate cancer cell lines DU145 and TRAMP-C2, respectively, autocrine IL-6 rendered cells unresponsive to STING agonists. While treatment with anti-IL-6 antibodies restored cGAMP responsiveness in DU145 cells, addition of recombinant IL-6 suppressed cGAMP-mediated upregulation of type I IFNs. In summary, our data suggest that cytosolic DNA activates the cGAS-STING signaling pathway. A functional STING is pivotal for eliciting an effective anticancer immune response. In most human cancer cell lines, however, STING signalling is inhibited. Since STING agonists are being evaluated in clinical trials, it is crucial to understand mechanisms that mediate STING unresponsiveness. We show that in tested prostate cancer cells, IL-6 signals contribute to unresponsiveness of STING and blocking of IL-6 can restore responsiveness to","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"20 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"126002071","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B157: Modulation of natural killer cell dysfunction in human bladder cancer","authors":"A. Farkas, F. Audenet, H. Anastos, M. Galsky, J. Sfakianos, N. Bhardwaj","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B157","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B157","url":null,"abstract":"T-cells undergo a progressive program of dysfunction termed “exhaustion” during chronic inflammatory pathologies such as cancer. T-cell exhaustion is characterized by diminished effector functionality and inefficient tumor immunosurveillance. However, it is unknown whether an analogous program undermines the contribution of innate lymphocytes like natural killer cells (NK) to tumor-surveillance, and what phenotype might define it. Therefore, we examined expression of inhibitory receptors and the corresponding functional potential of NK and T-cells present in peripheral blood mononuclear cells (PBMC) and tumor tissue of 59 individuals representative of the clinical spectrum of human bladder cancer (BC). PBMC and cell suspensions from freshly dissociated primary tumors were analyzed by flow cytometry to measure changes in the expression of the inhibitory receptors Tim-3, TIGIT, and PD-1, and to determine the composition of hematopoietic lineages in each tissue. NK effector function was assessed via activation with IL-2 or IL-15, followed by co-culture with Class I HLA-deficient targeT-cells to measure cytokine production and degranulation. Additionally, we determined the effect on NK cell function of ex vivo blockade of Tim-3 and TIGIT by the addition of monoclonal antibodies prior to functional assays. We found that NK cells significantly up-regulate expression of Tim-3 and TIGIT, but not PD-1, in both the PBMC and tumors of BC patients. T-cells demonstrate a similar pattern of expression. but with a far lower frequency of positive cells. The magnitude of Tim-3 expression by NK cells is a barometer of tumor invasiveness on cells in both PBMC and tumor tissue, while TIGIT is induced equivalently in BC patients independently of tumor stage. Importantly, both molecules are expressed at similar frequencies on NK cells isolated from blood or tumor, independent of the magnitude of overall expression, yet define NK with different functional potential. NK cells in PBMC from BC patients are functionally comparable to NK cells from healthy donors in their ability to produce IFNγ/TNFα and degranulate in response to targeT-cells, while tumor NK are refractory to both stimuli. NK cells from tumor tissue are not terminally exhausted as effector functions are restored after “resting” ex vivo prior to stimulation. Ex vivo blockade of Tim-3, but not TIGIT, enhances effector function in peripheral NK and T-cells from BC patients, but is ineffective for NK cells in tumor tissue, implicating suppressive factors specific to tumor in mediating NK dysfunction. Tim-3 blockade was most efficient in peripheral NK cells from BC patients that were activated with IL-15 versus IL-2, suggesting that local cytokine milieu can affect responsiveness to subsequent checkpoint inhibition. Overall, our data suggest that both NK and T-cells from patients with BC are enriched for expression of the shared inhibitory receptors Tim-3 and TIGIT, and that expression in the peripheral blood m","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"53 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"124934662","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Abstract B165: Investigating in vivo synergistic effect of checkpoint blockade and radiation therapy against chordomas in a humanized mouse model","authors":"Wataru Ishida, K. McCormick, A. Mahajan, E. Feldstein, M. Lim, J. Bruce, P. Canoll, S. Lo","doi":"10.1158/2326-6074.CRICIMTEATIAACR18-B165","DOIUrl":"https://doi.org/10.1158/2326-6074.CRICIMTEATIAACR18-B165","url":null,"abstract":"Introduction: With the advent of immunotherapy (IT) against various cancers, its applications to other cancers have been extensively investigated. However, it has been a challenge to apply IT to chordomas, due to lack of clinically translatable in vivo models. Currently, there are no well-established murine chordoma cell lines that can be injected to syngeneic mice or no transgenic mouse models that develop chordomas spontaneously, which would allow us to study the interaction between murine chordomas and murine immune cells. Hence, we aimed to develop a humanized mouse model, where human immune cells are engrafted into immunodeficient mice, to study the interaction between human immune system and human chordomas. We also sought to utilize it to investigate synergistic effect between IT and radiation therapy (RT) against chordoma. Materials and Methods: Fifteen 10-12-week-old NSG mice, which lack mouse T-cells, B cells, and NK cells as well as functional mouse macrophages, were sublethally (1.5Gy) irradiated and then implanted with fetal thymic tissue and CD34+ stem cells that had been harvested from a fetus, whose HLA-types were partially-matched with those of the U-CH1 chordoma cell line. Reconstitution of immune cells in NSG mice was confirmed eight weeks post-transplantation, and then each animal (15 humanized NSG mice and 12 naive NSG mice) was injected with U-CH1 cell suspension bilaterally and subcutaneously. Next, they were treated for 4 weeks as follows: A) control, isotype antibodies (Abs) injection (n=3), B) anti-human-PD-1 Abs (n=4, 10 mg/kg, 3 times/week for 4 weeks), C) RT + isotype Abs (n=3, unilaterally to the left-sided tumor, 8Gy x 4), D) anti-human-PD-1 Abs and RT (n=5), E) naive NSG mice (n=6, without the engraftment of human immune cells) + isotype, and F) naive NSG mice (n=6) + anti-human-PD-1 Abs. During and after the treatment, anti-tumor activities were monitored via tumor size, flow cytometry, qRT-PCR, and immunohistochemistry. Results: Eight weeks after stem cell engraftment, human peripheral blood mononuclear cells (PBMCs) of 43.8% among all PBMCs (human + mouse), human T-cells of 23.4% among human PBMCs, human CD8+ T-cells of 24.3% among human T-cells, and other lymphocytes such as B cells, macrophages, and NK cells were observed in peripheral blood of humanized mice via flow cytometry, which confirmed humanization. One week after the treatment, on the irradiated side, (D) demonstrated lowest tumor volume, highest number of human PBMCs, highest % of CD8+ human (cytotoxic) T-cells, highest % of CD45RO+CD4+ human (memory) T-cells, and lowest % of PD-1+CD8+ human (exhausted) T-cells in the tumors via flow cytometry, highest IFN-gamma in the tumors via qRT-PCR, and highest CD8+ human (cytotoxic) T-cells via immunohistochemistry, compared to the other five groups with statistical significance. Of note, on the nonirradiated side, a similar trend was observed with D) harboring the smallest tumor compared to the others (P=0.0","PeriodicalId":120683,"journal":{"name":"Other Topics","volume":"145 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2019-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"123780081","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}