bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A024: Predicting cancer survival with neo-epitope burden
Authors: Akers Nicholas K. , Schadt Eric , Losic Bojan
Published: November 2016

For many common cancers, survival and efficacy of state-of-the-art therapies are strongly predicted by the number of non-synonymous mutations. It is widely hypothesized that this association between mutations and survival is a result of mutations leading to neo-epitopes which in turn induce a robust anti-tumor immune response. It follows that accurately quantifying the number of neo-epitopes within a patient should better predict survival than that of non-synonymous mutations, however that has generally not been the case. Indeed, mutations and predicted neo-epitopes explain roughly the same survival variance, likely due to the difficulty of accurately predicting and prioritizing immunogenic neo-epitopes from mutations. We hypothesized that by integrating several data types and using state-of-the-art techniques for major histocompatibility complex (MHC) class I and II allele calling and epitope binding prediction, we could predict survival of cancer patients better than with mutation calls alone. We have developed a pipeline to characterize the potential immunogenicity of neo-epitopes. The pipeline uses copy-number variants and reads from whole-exome sequencing to assess the clonality of a given neo-epitope. MHC class I and class II allelotyping is performed, and expression of MHC alleles and mutations is determined using RNA-Seq. Similarity of epitopes to self-peptides is assessed by measuring similarity to predicted self-ligands, as well as by measuring the MHC affinity of wild-type versions of mutated epitopes. With this pipeline we were able to rapidly process the cancer genome atlas (TCGA) breast cancer (BRCA), non-small cell lung cancer (NSCLC), melanoma (SKCM), and pancreatic cancer (PAAD) datasets to infer neo-epitopes burden with high resolution. A linear model of survival as a function of mutations and their putative immunogenicity was constructed using these datasets via elastic net regularization, while modeling variance was controlled with k-fold cross validation. Our results confirm that the putatively immunogenic mutational burden is a highly significant term is such models. Finally, we quantify epitope response in these models by incorporating TCR sequence entropy as a proxy for clonal tumor infiltrating lymphocyte expansion.

Citation Format: Nicholas K. Akers, Eric Schadt, Bojan Losic. Predicting cancer survival with neo-epitope burden [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A024.
Abstract A028: Pre-clinical development of a first-in-class fully synthetic heat shock protein-based personalized cancer vaccine
Authors: Khattar Mithun , Tanne Antoine , Morin Benjamin , Wilson Nicholas , Uduman Mohamed , Zelin Justin , Stein Robert , Exley Mark , Castle John , Levey Daniel L.
Published: November 2016

Tumors accumulate spontaneous somatic mutations which can give rise to neo-epitopes (neoantigens) recognizable as non-self by T-cells. Advances in next-generation sequencing and bioinformatics make it possible to analyze individual patients' tumor genomes and predict immunogenic mutations, which can be synthesized as short peptides, RNA or DNA for use in personalized cancer vaccines. However, these approaches need optimization for safe and effective induction of anti-tumor T-cell responses. We addressed this challenge by complexing synthetic long peptides comprising predicted tumor neo-epitopes to recombinant Hsc70, creating personalized vaccine candidates, AutoSynVax™ (ASV™), administered with QS-21 Stimulon® adjuvant. The same platform comprising Hsc70 plus synthetic long viral peptides was previously validated in a Phase-2 clinical trial, demonstrating effective T-cell responses and reduced viral load in HSV-2+ subjects. ASV immunization is intended to facilitate antigen processing and presentation of neo-epitopes to T-cells, resulting in robust immune responses. The innate immune modulating properties of the Hsc70 protein as well as that of QS-21 Stimulon adjuvant are also under investigation at the vaccine injection site, in draining lymph node, and in cultures of antigen presenting cell populations exposed to the two agents. Finally, ASV is being explored in combination with various agonistic and antagonistic immune modulating monoclonal antibodies with the aim of increasing the duration of immunological memory and durability of tumor control. With an acceptable safety profile associated with Hsc70 and QS-21 Stimulon exposure in humans and demonstrated preclinical anti-tumor activity of ASV, we are poised to begin a first-in-human clinical trial by the first half of 2017.

Citation Format: Mithun Khattar, Antoine Tanne, Benjamin Morin, Nicholas Wilson, Mohamed Uduman, Justin Zelin, Robert Stein, Mark Exley, John Castle, Daniel L. Levey. Pre-clinical development of a first-in-class fully synthetic heat shock protein-based personalized cancer vaccine [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A028.
Abstract A036: Somato-germinomics antigens are immunogenic cancer stem cell antigens
Authors: Torigoe Toshihiko , Hirohashi Yoshihiko , Tsukahara Tomohide , Kanaseki Takayuki , Nakatsugawa Munehide , Kubo Terufumi , Vitaly Kochin
Published: November 2016

Introduction and Purposes: Cancer stem-like cells (CSCs) are the subpopulation of cancer cells that have stem-like phenotypes and high capacity for tumor initiation. 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 isolated CSCs by sphere formation assay or side population assay, and analyzed the immunopathological properties of CSCs derived from human solid cancers.Results: We could identify several genes that were characteristic to CSCs. Remarkably, some of them were expressed exclusively in testicles among normal adult organs and involved in spermatogenesis. siRNA-mediated knockdown of these genes caused decreased sphere-forming capacity and in vivo tumorigenicity, indicating that they were involved in the tumor initiation capacity of CSCs. They were highly immunogenic since specific cytotoxic T-cells (CTLs) were efficiently induced from peripheral blood lymphocytes of healthy volunteers as well as cancer patients. The CTLs exerted specific cytotoxicity against CSCs. We named them “Somato-germinomics antigens” in terms of the functioning antigens shared between somatic cancer cells and germ cells.Conclusion: Our data suggest that CSCs might utilize the specific machinery of germ stem cells for their tumor initiation and express immunogenic antigens. However, CSCs preferentially express PD-L1 on the cell surface, therefore escaping from immune surveillance. Based on these findings, we propose an immunotherapeutic strategy targeting CSCs.

Citation Format: Toshihiko Torigoe, Yoshihiko Hirohashi, Tomohide Tsukahara, Takayuki Kanaseki, Munehide Nakatsugawa, Terufumi Kubo, Kochin Vitaly. Somato-germinomics antigens are immunogenic cancer stem cell antigens [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A036.
Abstract A039: Accurate identification and prioritization of candidate neoantigens from cancer exome sequencing
Authors: White James , Simmons John , Angiuoli Sam , Sausen Mark , Jones Sian , Kann Lisa , Shukla Manish , Sevdali Maria , Velculescu Victor , Diaz Luis , Zhang Theresa
Published: November 2016

Somatic, nonsynonymous genetic alterations present in cancer can lead to the formation of novel protein sequences and thus production of immunogenic “non-self” neoantigens. Those neoantigens with sufficient expression will be processed and presented on MHC molecules, subsequently inducing a tumor-specific T cell response. Emerging data from clinical studies suggest that neoantigen load and composition is associated with the efficacy of some immunotherapies. As neoantigens play central a role in the cancer-immunity cycle, it is critical to identify the most potent immunogenic neoantigens effectively and accurately. Here we have leveraged highly accurate cancer whole exome (WES) analyses from FFPE tumor tissue with a state-of-the-art analysis protocol to identify and prioritize candidate neoantigens most likely to promote an immune response. ImmunoSelect-R utilizes somatic variants from WES to ensure detection of true somatic peptides and minimize false positives, and provides accurate HLA typing from whole exome sequencing data with >99.9% sensitivity and specificity. When applied to a set of experimentally validated neoantigens, ImmunoSelect correctly classified 18 out of 19 as strong neoantigen candidates, suggesting a sensitivity of greater than 90%. Moreover, in a small set of 10 patients, ImmunoSelect consistently ranked experimentally validated neoantigens within top 20% of all neoantigen candidates derived from whole exome sequencing. In summary, ImmunoSelect is able to identify and prioritize candidate neoantigens from cancer exome sequencing results effectively and accurately, enabling personalized cancer vaccine development, adoptive T-cell transfer, and prediction of response to checkpoint inhibitors

Citation Format: James White, John Simmons, Sam Angiuoli, Mark Sausen, Sian Jones, Lisa Kann, Manish Shukla, Maria Sevdali, Victor Velculescu, Luis Diaz, Theresa Zhang. Accurate identification and prioritization of candidate neoantigens from cancer exome sequencing [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A039.
Abstract A055: Systematic discovery of combinationimmunotherapy targets in vivo
Authors: Cartwright Adam N. , Tay Rong En , Wucherpfennig Kai W.
Published: November 2016

Immunotherapy has demonstrated remarkable responses in patients with metastatic tumors. Targeting the inhibitory CTLA-4 and PD-1 receptors expressed by T cells induces durable responses in a subset of cancer patients, including advanced melanoma. However, cancers, such as melanoma, remain largely resistant to many forms of immunotherapy, most likely due to exploitation of additional, and unidentified, negative feedback loops. It is widely appreciated that combination therapies will be required to extend the benefit of checkpoint blockade. Current approaches for discovery of combination therapies emphasize a 1 + 1 approach in which an active drug is tested in combination with an experimental agent. We hypothesize that many opportunities for synergistic combinations are missed with this strategy. We propose to develop a novel in vivo technique for discovery of combination therapies in the tumor microenvironment. Single-cell RNA-seq will be used to discover genes upregulated in dysfunctional T cells during PD-1 or CTLA-4 monotherapy in a mouse model of melanoma. We will then screen a library of identified targets in CD8 T cells for synergistic combinations using checkpoint blockade along with CRSIPR/Cas9 genome editing. Enrichment within the tumor of specific targets will then be further investigated to determine the most efficacious combinations for the treatment of melanoma.

Citation Format: Adam N. Cartwright, Rong En Tay, Kai W. Wucherpfennig. Systematic discovery of combinationimmunotherapy targets in vivo [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A055.
Abstract A057: CX3CR1 distinguishes three distinct migratory memory CD8 T cell subsets
Authors: Gerlach Carmen , Moseman E. Ashley , Loughhead Scott M. , Alvarez David , Zwijnenburg Anthonie J. , Waanders Lisette , Garg Rohit , Torre Juan C. de la , Andrian Ulrich H. von
Published: November 2016

CD8 T cells are crucial for the protection against tumors and intracellular pathogens via their ability to generate long-term memory. Memory T cells (TMem) are however not a homogenous population. Traditionally, blood-borne TMem have been sub-divided into central memory (TCM) and effector memory (TEM) cells based on different migration behaviors and cytotoxic potential.

We recently discovered the existence of a novel subset of CD8 TMem that has unique migratory properties and a superior homeostatic self-renewal capacity. The hitherto unrecognized memory CD8 T cell subset is characterized by intermediate expression levels of CX3CR1, while classical TCM were CX3CR1−, and TEM were CX3CR1high. The fraction of CX3CR1int cells among antigen-experienced TMem was remarkably stable over the course of a year, while TEM gradually declined and TCM increased to be the largest subset by 10 months. Interestingly, CX3CR1int TMem had the highest proliferation rate of the three TMem subsets. While TCM and TEM were phenotypically stable over >10 weeks at steady state, CX3CR1int TMem not only self-renewed, but also gave rise to bonafide TCM. Consequently, CX3CR1int TMem contributed to the gradual increase in TCM.

The newly identified CX3CR1int TMem displayed unique migratory properties. Unlike CX3CR1high TEM, CX3CR1int gradually acquired lymph node homing capability. Moreover, parabiosis experiments and analysis of recirculating memory cells in thoracic duct lymph revealed that CX3CR1int TMem, not TEM, are the predominant T cell subset surveying non-lymphoid peripheral tissues.

Supported by a postdoctoral fellowship of the Cancer Research Institute Irvington Fellowship Program and a Rubicon fellowship (Netherlands Organization for Scientific Research, NWO) to CG, NIH T32 Training Grant in Hematology 5T32-HL07623-20 to EAM, NIH F31 grant CA171339 to SML, NIH T32 grant HL066987 to DA, the Ragon Institute of MGH, MIT and Harvard and NIH/NIAID RO1 AI069259, PO1 AI078897 and PO1 AI112521 to UHvA.

Citation Format: Carmen Gerlach, E. Ashley Moseman, Scott M. Loughhead, David Alvarez, Anthonie J. Zwijnenburg, Lisette Waanders, Rohit Garg, Juan C. de la Torre, Ulrich H. von Andrian. CX3CR1 distinguishes three distinct migratory memory CD8 T cell subsets [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A057.
Abstract A058: Selective targeting of T regulatory cells by a TCR-mimic monoclonal antibody specific for foxp3-derived epitopes
Authors: Dao Tao , Jarvis Casey , Scott Andrew C. , Korontsvit Tatyana , Zakhaleva Victoria , Pankov Dmitry , Guerrerio Manuel Direito de Morais , Mathias Melissa , Cheng Neal , Liu Cheng , Scheinberg David A.
Published: November 2016

Immunosuppression by tumor-induced regulatory T (Treg) cells present major obstacles for successful immunotherapy. Treg expansion and its negative prognostic impact represent a general phenomenon seen in multiple types of cancer. Therefore, developing strategies for Treg depletion could enhance the reactivation of immune responses against the malignant tumor cells. However, the strategies of depleting Tregs have been hindered by the lack of specificity, which also leads to the depletion of anti-tumor effector cells. The transcription factor forkhead box p3 (FoxP3) is selectively expressed in and is essential to the suppressive function of Treg cells. FoxP3 thus would be an appealing target for eliminating Treg cells. However, Foxp3 is an undruggable intracellular protein. Here, we took a novel and unconventional approach to target Foxp3 by using a T cell receptor mimic (TCRm) mAb, which recognizes a human Foxp3-derived CD8 T cell epitope, presented by HLA-A*02:01 molecule. We show that the Foxp3 mAb specifically bound to CD4+CD25hiCD127lo Foxp3+ Treg cells from HLA-A0201 positive donors, and tumor cell lines co-expressing Foxp3 and HLA-A*02;01 molecule. Both afucosylated Fc enhanced human IgG1 and bispecific T cell engager formats of the Foxp3 mAb are able to kill in vitro-generated Treg clones from HLA-A0201+ donors and “Treg-like” cutaneous lymphoma cells (HLA-A*02:01+) that have a high level expression of CD4, CD25 and Fox3+. FoxP3-targeting antibodies could potentially be a novel approach in cancer immunotherapy by overcoming immunosuppression caused by Tregs and tumor cells expressing Foxp3.

Citation Format: Tao Dao, Casey Jarvis, Andrew C. Scott, Tatyana Korontsvit, Victoria Zakhaleva, Dmitry Pankov, Manuel Direito de Morais Guerrerio, Melissa Mathias, Neal Cheng, Cheng Liu, David A. Scheinberg. Selective targeting of T regulatory cells by a TCR-mimic monoclonal antibody specific for foxp3-derived epitopes [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A058.
Abstract A063: Dissecting adipose tissue Treg differentiation and function in metabolism and obesity-associated cancer using TCR transgenic mice
Authors: Li Chaoran , Benoist Christophe , Mathis Diane
Published: November 2016

Recent epidemiological and animal model studies indicate that overweight and obese individuals have increased risk not only of type-2 diabetes and cardiovascular diseases but also of various types of cancer. It was suggested that chronic, low-grade inflammation of the adipose tissue, and eventually systemically, is one of the major causes for obesity-associated metabolic abnormalities and cancer, but the trigger, progression and regulation of obesity-induced inflammation and how these processes might influence the development of cancer remain poorly understood. Our laboratory previously discovered a unique population of Foxp3+ CD4+ regulatory T cells (Tregs), with a distinct transcriptome and clonally expanded T cell receptor (TCR) repertoire, residing in the visceral adipose tissue (VAT) of mice and humans. These cells are critical for regulating local inflammation and maintaining systemic insulin sensitivity, but whether they regulate the development of obesity- associated cancers is currently unknown. Moreover, we are still at the early stages of an integrative understanding of the differentiation, trafficking, and expansion of this unique population of VAT cells. Attempting to fill some of these knowledge-gaps, we developed a VAT Treg TCR transgenic (Tg) mouse line, in which Tregs preferentially accumulate in VAT upon recognition of a currently unknown antigen. With this unique tool, we elucidated the life history of VAT Tregs, determined when and where they accumulate and acquired the unique phenotype, identified factors that are important for their homeostasis, and demonstrated their function in suppressing insulin resistance. We are currently in the process of elucidating their function in controlling the obesity- promoted liver inflammation and tumorigenesis. This study will provide foundations for Treg-based immunotherapy against the metabolic syndrome and obesity-associated cancer, while pointing to new cellular and molecular pathways that may constitute potential therapeutic targets.

Citation Format: Chaoran Li, Christophe Benoist, Diane Mathis. Dissecting adipose tissue Treg differentiation and function in metabolism and obesity-associated cancer using TCR transgenic mice [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A063.
Abstract A066: NF-kB restricts inflammasome activation via elimination of damaged mitochondria
Authors: Zhong Zhenyu , Karin Michael
Published: November 2016

NF-κB, a key activator of inflammation primes the NLRP3-inflammasome for activation by inducing pro-IL-1β and NLRP3 expression. NF-κB, however, also prevents excessive inflammation and restrains NLRP3-inflammasome activation through a poorly defined mechanism. We now show that NF-κB exerts its anti-inflammatory activity by inducing delayed accumulation of the autophagy receptor p62/SQSTM1. External NLRP3-activating stimuli trigger a form of mitochondrial (mt) damage that is caspase-1- and NLRP3-independent and causes release of direct NLRP3-inflammasome activators, including mtDNA and mtROS. Damaged mitochondria undergo Parkin-dependent ubiquitin conjugation and are specifically recognized by p62, which induces their mitophagic clearance. Macrophage-specific p62 ablation causes pronounced accumulation of damaged mitochondria and excessive IL-1β-dependent inflammation and enhances macrophage death. Therefore, the “NF-κB-p62-mitophagy” pathway is a macrophage-intrinsic regulatory loop through which NF-κB restrains its own inflammation-promoting activity and orchestrates a self-limiting host response that maintains homeostasis and favors tissue repair.

Citation Format: Zhenyu Zhong, Michael Karin. NF-kB restricts inflammasome activation via elimination of damaged mitochondria [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A066.
Abstract A072: Glucose metabolism and O-linked GlcNAcylation in the tissue repair function of Treg cells
Authors: Hu Wei , Arpaia Nicholas , Green Jesse A. , Hendrickson Ronald C. , Rudensky Alexander Y.
Published: November 2016

Regulatory T (Treg) cells dampen immune responses to avoid excessive, deleterious inflammation in the context of autoimmunity, allergy, and infection. Treg cells also impede anti-tumor immunity. Therefore, modulation of Treg cell activity is of great therapeutic potential for treating many debilitating inflammatory diseases and cancer. Recent studies have suggested that in addition to their prominent immunosuppressive function, Treg cells have a distinct and non-redundant role in tissue maintenance and remodeling. These Treg cells exhibit a unique transcriptional signature represented by high expression of the EGF family member amphiregulin (Areg). Importantly, tumor infiltrating Treg cells highly express Areg. Since Areg is known to exert pleiotropic oncogenic activities, Areg producing Treg cells may be crucial for establishing the tumor microenvironment. It has been increasingly appreciated that lymphocyte activation and differentiation are coupled to specific metabolic pathways. Metabolic reprogramming is dictated by cytokine and growth factor signals, as well as the availability of nutrients. In addition, metabolic products provide substrates that can alter the functional status of a cell through posttranslational modifications (PTMs). Among the various nutrients, glucose is a key metabolic substrate for T cells. Aerobic glycolysis in activated T cells is required for the acquisition of full effector function. Treg cells in the secondary lymphoid organs and the tissue have distinct glucose availability and metabolic needs. We thus hypothesized that glucose sensing and glucose metabolism play essential roles in shaping the unique phenotype and function of tissue resident Treg cells. O-linked GlcNAcylation is a PTM dynamically regulated by glucose levels and controls the function of many proteins, including transcription factors that play key roles in T cell activation such as NF-κB, CREB, and NFAT. To understand the mechanism by which glucose metabolism controls the function of tissue-resident Treg cells, we began to characterize protein O-GlcNAcylation in Treg cells, and found that both human and mouse Foxp3, the master regulator transcription factor of Treg cells, undergoes O-GlcNAcylation. We are in the process of mapping the site of O-GlcNAcylation of Foxp3, and understanding the physiological significance of this PTM.

Citation Format: Wei Hu, Nicholas Arpaia, Jesse A. Green, Ronald C. Hendrickson, Alexander Y. Rudensky. Glucose metabolism and O-linked GlcNAcylation in the tissue repair function of Treg cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A072.
Abstract A077: Identifying novel effectors of the gut microbiota that modulate cancer cell killing by CTLs using functional metagenomics
Authors: Lobel Lior , Garret Wendy
Published: November 2016

The immune system, which has close interactions with the microbiota, plays an anti-tumorigenic role by surveying for and killing cancer cells. In this regard, recent studies found the microbiota to be essential for the success of cancer immunotherapy treatments in mice, while the bacterial effectors remain unknown. We propose an innovative high throughput screening system to isolate microbiota-derived effectors that modulate the ability of immune cells to kill cancer cells. We utilize the mouse CT26 colon carcinoma cell line as target cells and the GSW11 specific CCD2Z T cell hybridoma cell line as the effector cells. Cytotoxicity and T cell activation assays, measured by LDH release and LacZ expression, respectively, were calibrated to both positive and negative controls and in response to an array of perturbations. Bacterial DNA from mouse colon tumors will serve for the generation of metagenomics libraries. We then plan to screen ∼ 30,000 clones and characterize the cellular and immune responses of identified effectors both in tissue cultures and in animals. Finally, we hope that our future results could be taken to the clinic and serve for diagnosis, prognosis and therapeutics.

Citation Format: Lior Lobel, Wendy Garret. Identifying novel effectors of the gut microbiota that modulate cancer cell killing by CTLs using functional metagenomics [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A077.
Abstract A080: Balance of commensal bacteria specific Th17 and RORγt+ Treg cells in intestinal homeostasis and inflammation
Authors: Xu Mo , Yang Yi , Pokrovskii Maria , Galan Carolina , Littman Dan R.
Published: November 2016

A number of Helicobacter species, including Helicobacter hepaticus, have been isolated as the etiological agents of ulcerative inflammatory bowel disease and cancer in their respective hosts. H. hepaticus induces severe Th17-Th1 mediated colitis in a number of immuno-compromised mouse strains, but does not usually cause disease in most wild-type mouse strains. However, how the inflammatory response is carefully contained to be harmless under homeostatic conditions and how perturbations lead to pathogenicity remain elusive questions. To understand this, we developed new research tools, including TCR transgenic mice, as well as MHCII tetramers to interrogate the regulation of H. hepaticus-specific CD4+ T cell function. Interestingly, in contrast to Segmented Filamentous Bacteria (SFB), which induces Th17 cells in the small intestinal lamina propria (SILP) of both IL10+/− and IL-10−/− mice, H. hepaticus mainly induces RORgt+ Treg cells in the large intestinal lamina propria (LILP) of IL-10-sufficient mice, but induces a Th17-Th1 response in the absence of IL-10. We propose that the balance between self/commensal antigen-induced RORgt+ Treg and Th17 cells may be a general strategy to control autoimmune inflammation.

Note: This abstract was not presented at the conference.

Citation Format: Mo Xu, Yi Yang, Maria Pokrovskii, Carolina Galan, Dan R. Littman. Balance of commensal bacteria specific Th17 and RORγt+ Treg cells in intestinal homeostasis and inflammation [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A080.
Abstract A087: Quantifying the landscape of immunostimulatory tumoral RNA
Authors: Solovyov Alexander , Tanne Antoine , Muniz Luciana , Cocco Simona , Monasson Remi , Levine Arnold , Ting David T. , Bhardwaj Nina , Greenbaum Benjamin
Published: November 2016

Recent studies demonstrate an unexpected connection between aberrant transcription of noncoding RNA in tumors and innate immune system activation in the tumor microenvironment. Such RNA is often of unknown function and may consist of typically silenced interspersed elements, satellite repeats, and endogenous retroviruses. For instance, satellite RNA from the pericentromere (particularly HSATII) is abundantly transcribed in several solid tumors - such as pancreatic ductal adenocarcinoma - yet it is virtually silent in normal tissue. The genomic DNA repetitive regions this RNA is transcribed from frequently expand during tumorigenesis. Using novel quantitative methods, we have shown that a set of such repetitive elements, abundantly expressed in tumors, display sequence patterns typically associated with viruses. We therefore predicted they are immunogenic, particularly HSATII. In a novel, theory-experiment collaboration between the laboratories of Professors Benjamin Greenbaum and Nina Bhardwaj, the most significant set of these RNA have been validated as immunostimulatory (HSATII and murine GSAT), capable of activating antigen presenting cells - HSATII stimulated production of IL-6, IL-12 and TNFalpha (Tanne, et al., PNAS, 2015).

At the same time a set of recent papers has shown that ERV transcription may be a predictor of immunotherapy response in melanoma. Hence, it is critical to profile key immunostimulatory endogenous RNA in the tumor microenviroment, understand which immune pathways different sets of such RNA activate, and assess the link between the specific pathways activated, prognosis, and immunotherapy. We further profile the landscape of activation and expression for endogenous elements. We have currently demonstrated a key set of noncoding RNAs preferentially expressed in cancer cells have sequence features that are immunostimulatory in humans, and have characterized the range and breadth of the expression of such elements in several solid tumors. We present several new results on the topic and the potential consequences of the aberrant expression of endogenous RNA that mimic pathogen features in cancer.

Citation Format: Alexander Solovyov, Antoine Tanne, Luciana Muniz, Simona Cocco, Remi Monasson, Arnold Levine, David T. Ting, Nina Bhardwaj, Benjamin Greenbaum. Quantifying the landscape of immunostimulatory tumoral RNA [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A087.
Abstract A089: Multiparametric immunofluorescence analysis of the tumor microenvironment using CODEX
Authors: Kennedy-Darling Julia , Nolan Garry P. , Goltsev Yury , Samusik Nikolay
Published: November 2016

The tumor microenvironment plays a critical role in cancer progression and has implications for the efficacy of various cancer immunotherapy treatment options. Immune infiltrates within the tumor microenvironment can correlate with both positive and negative outcomes, depending upon the both the type of cancer as well as infiltrating immune cell(s). These analyses are typically performed using standard immunofluorescence and immunohistochemistry assays where no more than four simultaneous parameters can be visualized on the same tissue. Unfortunately, these tools cannot fully characterize the complexity of the tumor microenvironment due to the inherent limitations of fluorophore spectral overlap. In order to identify each type of immune and tumor cell within a single tissue, at least 40 parameters need to be measured simultaneously. We have developed a multiparametric immunofluorescence technology, entitled CODEX (Co-Detection by IndEXing), which utilizes unique DNA tags as a means of iteratively measuring more than 40 parameters within the same tissue. More than 40 human antibodies have been validated using this approach, including numerous immune markers, checkpoint ligands, tumor markers and cellular activity markers. We are currently analyzing tissue sample from patients with lung cancer. By measuring nearly 50 simultaneous markers within the same tissue, CODEX has the potential to greatly enhance our knowledge of the tumor microenvironment and more accurately define immune infiltrates at the single-cell level.

Citation Format: Julia Kennedy-Darling, Garry P. Nolan, Yury Goltsev, Nikolay Samusik. Multiparametric immunofluorescence analysis of the tumor microenvironment using CODEX [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A089.
Abstract A093: Impact of antigen affinity on T cell dysfunction in solid tumors
Authors: Shakiba Mojdeh , Philip Mary , Schietinger Andrea
Published: November 2016

Although tumor-specific CD8 T cells are found in human solid tumors, cancers progress, indicating that these T cells are dysfunctional. When naïve CD8 T cells encounter antigen in an inflammatory context (e.g. acute infection), a cell-intrinsic program is initiated that drives T cells to expand, produce effector cytokines, and differentiate into effector and memory T cells. In contrast, dysfunctional CD8 T cells in solid tumors express high levels of inhibitory receptors and fail to execute effector functions; however, the regulatory mechanisms underlying tumor-specific T cell dysfunction remain poorly defined. T cell-mediated immune responses are triggered by T cell receptor (TCR) binding to peptide-major histocompatibility complex (pMHC) on the surface of antigen presenting cells. The affinity of TCR:pMHC interaction is a critical determinant of CD8 T cell expansion and effector function in acute infections; while both low- and high-affinity antigens can activate naïve antigen-specific CD8 T cells, T cells with high-affinity TCR:pMHC interactions generally show superior effector function. However, little is known about how tumor antigen affinity impacts the activation of T cells, the induction of T cell dysfunction in progressing tumors, and T cell susceptibility to immunotherapeutic reprogramming. To investigate the role of TCR:pMHC affinity in T cell differentiation in progressing tumors, we generated tumor cell lines expressing altered peptide ligands (APL) derived from SV40 large T antigen epitope I (SV40-I). These APLs are recognized by SV40-I-specific transgenic CD8 T cells (TCRSV40-I) with varying functional avidity. We found that tumor-infiltrating TCRSV40-I encountering low- and high-affinity tumor antigens were equally activated and displayed similar expression levels of inhibitory receptors, such as PD1 and Lag3, suggesting that even very weak TCR ligations can induce a typical “exhaustion” phenotype. Strikingly, while high-affinity TCR:pMHC interactions led to complete loss of interferon gamma and tumor necrosis factor alpha production, T cells with low-affinity interactions remained functional and capable of producing high levels of these effector cytokines suggesting that contrary to our expectations, tumor-specific T cells with high-affinity TCR:pMHC interactions enter a profound state of dysfunction. Future experiments will test the impact of tumor antigen affinity on the efficacy of therapeutic reprogramming including checkpoint blockade, which will have important implications for immunotherapy.

Citation Format: Mojdeh Shakiba, Mary Philip, Andrea Schietinger. Impact of antigen affinity on T cell dysfunction in solid tumors [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A093.
Abstract A109: Immune profiling of tumor infiltrating lymphocyte subsets, myeloid cells and HPV status of HNSCC biopsies using multiplex immunofluorescence
Authors: Levin Matt , Lingen Mark , Schwartz David , Snyder Helen
Published: November 2016

Head and neck squamous cell carcinoma (HNSCC) has both human papilloma virus (HPV) positive and negative etiologies. It is one of the growing number of tumors for which an anti-tumor immune response is implicated to play a role in the course of disease as shown by responsiveness to immune-modulation using PD-1/PD-L1 inhibitors. Profiling of the tumor microenvironment including specific infiltrating lymphocyte subsets, antigen presenting cells, expression of regulatory molecules on both tumor and immune cells, and spatial relationships between cells could lead to predictive signatures for responsiveness to both conventional and immune modulatory therapies. However, such profiling has previously been hindered by the need to identify such cells “in-situ” in tissue sections using multiple markers per cell, and limitations in available technology. The current study demonstrates Cell IDx's novel UltraPlex technology, based on a simple two step protocol using hapten labeled primary antibody cocktails followed by fluor labeled anti-hapten secondary cocktails, allowing simultaneous detection of 4 markers on the same cell. Analysis of HNSCC positive and negative tissue microarrays combining UltraPlex detection of multiple markers per cell together with serial sections stained with individual phenotyping panels demonstrates a powerful technique for 12-16 plex analysis. This permits HPV status determination, immune subset profiling, quantitative marker expression, relative cellular localization and potential for correlative signature determination associated with disease outcome and response to treatment.

Citation Format: Matt Levin, Mark Lingen, David Schwartz, Helen Snyder. Immune profiling of tumor infiltrating lymphocyte subsets, myeloid cells and HPV status of HNSCC biopsies using multiplex immunofluorescence [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A109.
Abstract A112: Manipulating tumor microenvironment for immunotherapy
Authors: Tang Haidong , Qiu Xiangyan , Wang Yang , Fu Yang-Xin
Published: November 2016

An established tumor is a complex tissue composed of tumor cells, stromal cells, inflammatory cells, vasculatures, and extracellular matrices, all of which are defined together as the tumor microenvironment. Successful tumor control by immunotherapy requires activation of the immune system, expansion of the effector cells, infiltration of activated effector cells to the tumor tissue, and destruction of the tumor cells. Our previous studies have showed that anti-tumor effect of current tumor immunotherapies, such as checkpoint blockade, relies on a significant number of spontaneous tumor infiltrating lymphocytes. However, established tumors often inhibit peripheral lymphocytes entering tumor tissues for tumor destruction. Inflammatory mediators, especially cytokines, chemokines, and adhesion molecules, play important roles in the recruitment and activation of various types of innate and adaptive immune cells. As a proof-of-concept study, we targeted tumor tissues with LIGHT, a member of the tumor necrosis factor super family. Our data suggested that targeting tumor with LIGHT is able to induce the production of several cytokines/chemokines, which recruit T cells into tumor microenvironment, leading to tumor regression. Furthermore, combination therapy with LIGHT is able to overcome tumor resistance to checkpoint blockade. Thus, these data suggested that manipulating tumor microenvironment with cytokines/chemokines that are able to recruit effector cells represents a promising strategy for tumor immunotherapy. To better understand the role of individual cytokine/chemokine, we utilized chemokine array to systemically analyze tumor microenvironments. By comparing chemokine profiles from immunotherapy-sensitive and -insensitive tumors, we found a significant difference which might be responsible for their sensitivity to immunotherapies. Sensitive tumor is characterized with a higher level of chemokines associated with lymphoid cell trafficking, such as CXCL9 and CCL22. In contrast, insensitive tumor is characterized by a higher levels of chemokines associated with myeloid cell, such as MCP-1 and CCL11. The contributions and their therapeutic potentials of these cytokines/chemokines will be studied in the next step.

Citation Format: Haidong Tang, Xiangyan Qiu, Yang Wang, Yang-Xin Fu. Manipulating tumor microenvironment for immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A112.
Abstract A118: CD103+/PD-1+ T-cells identify a subset of triple-negative breast cancer eligible for targeted checkpoint inhibition
Authors: Bottai Giulia , Roncalli Massimo , Santarpia Libero
Published: November 2016

The presence of tumor-infiltrating lymphocytes (TILs) is a prognostic factor in triple-negative breast cancer (TNBC). Specifically, cytotoxic CD8+ cells are associated with favorable outcome in TNBC. Recently, the integrin CD103 has been suggested as a potential prognostic marker in breast cancer. In this study, we aimed to evaluate the functional status and clinical relevance of CD8+ and CD103+ TILs in TNBC.

A retrospective cohort of 210 formalin-fixed, paraffin-embedded tissues from patients with primary invasive ductal TNBC was assessed for the presence of CD8, CD103 and the checkpoint receptor PD-1 by immunohistochemistry assay. Whole tumor slides were assessed. Cases where ≥5% of TILs expressed CD103 or PD-1 were considered positive. Statistical analyses were performed using Spearman's correlation, Kaplan-Meier and Cox regression analyses.

We found that CD103+ cells mostly co-expressed the CD8 marker, and were preferentially distributed within tumor epithelium. No consistent correlation was found between the presence of CD103+ lymphocytes and the expression of its ligand E-cadherin by TNBC cells (rs = 0.365). CD8+ lymphocytes were consistently associated with better relapse-free survival (RFS) and overall survival (OS) in both univariate (HR = 0.61; P = 1.06E-02 for RFS; HR = 0.59; P = 1.93E-02 for OS) and multivariate analysis (HR = 0.65; P = 2.45E-02 for RFS; HR = 0.63; P = 3.96E-02 for OS). The presence of CD103+ lymphocytes significantly correlated with prolonged RFS and OS in univariate analysis only (HR = 0.82; P = 4.12E-02 for RFS; HR = 0.85; P = 4.93E-02 for OS), and a trend for longer RFS was also observed in univariate analysis for PD-1 (HR = 0.71; P = 5.22E-02). Interestingly, a subset of TNBC showed co-expression of CD103+/PD-1+ in lymphocytes localized to the intraepithelial areas of the tumor.

The expression of CD103 on CD8+ T cells in direct contact with cancer cells may identify a subpopulation of cells with potent cytolytic activity. The interaction between CD103+ cytotoxic lymphocytes and TNBC cells is mediated by different mechanisms rather than the binding to E-cadherin. Even though CD103 has a role in mediating an effective anti-tumor immune response, its presence alone may not be sufficient to impact the outcome of TNBC patients. Immunomodulatory therapies may be useful to boost the anti-tumor activity of potentially quiescent CD103+/PD-1+ cells in a subgroup of TNBC patients.

Citation Format: Giulia Bottai, Massimo Roncalli, Libero Santarpia. CD103+/PD-1+ T-cells identify a subset of triple-negative breast cancer eligible for targeted checkpoint inhibition [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A118.
Abstract A136: p16INK4a status of primary tumor modulates local immune response in vulvar squamous cell carcinoma
Authors: Sznurkowski Jacek J. , Zawrocki Anton
Published: November 2016

Background: The p16INK4a is not a surrogate marker for high-risk human papilloma virus genotypes (hr)HPV-DNA) and was suggested to indicate better prognosis of vulvar squamous carcinoma (vSCC). Our recent study confirmed substantial mismatch between p16ink4a and (hr)HPV-status as well as prognostic significance exclusively for p16INK4a-overexpression in cohort of 85 patients. Aim: To compare the infiltration of tumor infiltrating lymphocytes (TILs) and to assess their prognostic significance in cases having tumors with opposed p16INK4a and (hr)HPV-status.

Methods: TILs (CD8+, CD4+, FOXP3+, CD68+, CD56+ and GzB+ cells) were calculated in 85 vSCCs with previously defined p16INK4a and (hr)HPV-status. Number of intraepithelial (IE) TIL-subtypes were compared between tumors with different p16INK4a and (hr)HPV-status separately. Survival analyses included the Kaplan-Meier method, log-rank test and Cox proportional hazards model.

Results: p16INK4a-negative tumors were infiltrated more intensely by CD8+, CD4+ and GZB+ cells than p16INK4a-positive vSCCs (p = 0.032, p = 0.016 and p = 0.007 respectively). (hr)HPV-status did not impact the infiltration of TILs. Median follow up was 89.20 months (range 1.7-189.5). High (IE)CD4+ infiltrates were prognostic in general cohort and in p16INK4a-positive cases (p = 0.032 and p = 0.039 respectively). High (IE)CD56+ infiltrates were correlated with prognosis in p16INK4a-positive and (hr)HPV-positive cases (p = 0.013 and p = 0.0458 respectively) while high (IE)CD68+ infiltrates were inversely correlated with survival in cases with p16INK4a-negative and (hr)HPV-negative tumors (p = 0.018 and p = 0.0441 respectively).

Conclusion: p16INK4a-status modulates local immune surveillance as represented by TILs. Immunologic effects contributing to clinical outcome might depend on p16INK4a-overexpression.

Citation Format: Jacek J. Sznurkowski, Anton Zawrocki. p16INK4a status of primary tumor modulates local immune response in vulvar squamous cell carcinoma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A136.