bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract B132: A new concept of cancer immunotherapy by targeting pancreatic ductal adenocarcinoma through a childhood recall antigen
Authors: Selvanesan Benson C. , Chandra Dinesh , Quispe Wilber , Patel Ankur , Meena Kiran , Libutti Steven K. , Yuan Ziqiang , Chuy Jennifer , Gravekamp Claudia
Published: February 2019

Patients with pancreatic ductal adenocarcinoma (PDAC) have a five-year survival of only 6%. Cancer immunotherapy has shown promising results for metastatic cancer but only in 20% of the patients. Our lab has developed a new concept of immunotherapy using an attenuated non-pathogenic bacterium Listeria monocytogenes for the selective delivery of a highly immunogenic recall antigen tetanus toxoid (TT) into tumor cells of pancreatic tumors and metastases. Most individuals have been exposed to TT during childhood vaccinations and memory T-cells to TT circulate in blood for live. These memory T-cells to TT can be reactivated by Listeria-TT at any time in life and will now destroy the infected tumor cells. Low doses of gemcitabine (GEM) were added to further improve T-cell responses. Panc-02 and KPC mice were immunized with the “human TT vaccine” to generate memory T-cells before development of the pancreatic cancer. After cancer development, mice were treated with Listeria-TT and GEM, and tumors (weight) and metastases (number) were analyzed by eye in the Panc-02 mice, and by SUVmax (measurement of tumor growth) in the KPC mice. T-cell responses in the spleen were analyzed by flow cytometry and ELISPOT, and KPC tumors were analyzed by RNAseq. Listeria-TT and GEM nearly completely eliminated both early and advanced pancreatic cancer in Panc-02 and KPC mice, in correlation with abundant CD4 and CD8 T-cell responses producing perforin, granzyme B, or IFNγ, in vivo or in vitro, respectively. RNAseq analysis of “treated” KPC tumors showed a strong influx of CD4 and CD8 T-cells, upregulation of multiple granzymes, perforin, co-stimulatory molecules, and cell death pathways. These results suggest the multiple synergistic effects of Listeria-TT and GEM on pancreatic cancer. This novel treatment modality of Listeria-TT and GEM warrants further investigation in a clinical setting.

Citation Format: Benson C. Selvanesan, Dinesh Chandra, Wilber Quispe, Ankur Patel, Kiran Meena, Steven K. Libutti, Ziqiang Yuan, Jennifer Chuy, Claudia Gravekamp. A new concept of cancer immunotherapy by targeting pancreatic ductal adenocarcinoma through a childhood recall antigen [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 B132.
Abstract B149: Real-time analysis of cell membrane protein and virus internalization using site-specific conjugation of protease-sensitive probes
Authors: Cheloha Ross W. , Li Zeyang , Bousbaine Djenet , Woodham Andrew , Perrin Priscillia , Volaric Jana , Ploegh Hidde L.
Published: February 2019

Reagents that label specific subsets of cells are broadly useful for the treatment of cancer and modulation of cells of the immune system. Labeling is often achieved through the identification and characterization of proteins specifically expressed on selected cells but not others. Knowledge of cell membrane protein routing dynamics can be leveraged to efficiently deliver cytotoxic or immune stimulating payloads. The most common method for monitoring internalization relies on labeling proteins with antibodies modified with a fluorophore or other tag that can also be used to report on whether the protein of interest has been internalized. This approach requires indirect methods, such as multiple rounds of cell staining, to differentiate extracellular protein from protein that has been internalized and recycled to the cell surface. Here we report a method for the characterization of protein internalization in real time through the sortase-mediated, site-specific labeling of single domain antibodies or viral proteins with a newly developed, cathepsin-sensitive quenched-fluorophore probe. This approach allows quantitative measurement of the movement of proteins into protease-containing endosomes in real time in live cells. This method revealed variation in the rate of internalization for different cell surface receptors and allowed for kinetic characterization of influenza virus internalization. These findings help to explain the utility of certain single domain antibody-antigen conjugates for inducing humoral immune responses. The tools and methods described here should be useful for the identification of proteins expressed on target cells that are ideal for antibody-mediated drug delivery or for promotion of specific types of immune responses.

Citation Format: Ross W. Cheloha, Zeyang Li, Djenet Bousbaine, Andrew Woodham, Priscillia Perrin, Jana Volaric, Hidde L. Ploegh. Real-time analysis of cell membrane protein and virus internalization using site-specific conjugation of protease-sensitive probes [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 B149.
Abstract B156: Characterization and role of CCR8+ regulatory T-cells in mouse models of malignant melanoma
Authors: Edwards Andrew K. , Skobe Mihaela , Rogic Anita , Bosenberg Marcus
Published: February 2019

Melanoma cells disseminate through lymphatic vasculature into the regional lymph nodes early in disease progression. We found that a large subset of metastatic melanomas express chemokine receptor CCR8. Its principal ligand, CCL1, is constitutively expressed by lymphatic endothelial cells in the lymph node and is further upregulated by inflammation. The CCL1-CCR8 axis is an important checkpoint for melanoma lymph node metastasis, as an inhibition of CCR8 leads to arrest of melanoma cells in collecting lymphatic vessels and prevents lymph node metastasis. Among immune cells, CCR8 is predominantly expressed by regulatory T-cells (Treg) and by activated Th2 cells, and has been implicated mainly in allergic inflammation. Here we characterized CCR8+ immune cells and examined the role of CCR8 in mouse models of melanoma. Using inducible, genetically engineered (Tyr::CreER;BrafCAPtenloxPCtnnb1loxex3) and syngeneic mouse models of melanoma (B16F10) we characterized CCR8 expression on different immune cell subsets in primary tumors and in sentinel lymph nodes by flow cytometry. In primary tumors, CCR8 was exclusively expressed by CD4+ T-cells, with the highest percentage being FOXP3+CCR8+ Tregs. Increased numbers of CD4+FOXP3+CCR8+ Tregs were found in sentinel lymph nodes with metastases, compared to non-tumor-draining lymph nodes and lymph nodes from tumor-naïve mice. This also corresponded with an increase in the total number of FOXP3+ T-regs in sentinel lymph nodes, compared to non-tumor draining lymph nodes. We then evaluated the role of CCR8 on B16F10 tumor growth. Tumor growth was significantly reduced in CCR8-/- mice compared to WT mice when anti-tumor immunity was stimulated with Poly(I:C). However, B16F10 tumor growth did not differ between untreated wild-type (WT) and CCR8 -/- mice. These results suggest that targeting CCR8+ Tregs may increase antitumor immune response. Further studies are required to determine the mechanism by which CCR8+ immune cells facilitate melanoma growth and to explore CCR8 as a therapeutic target in melanoma immunotherapy.

Citation Format: Andrew K. Edwards, Mihaela Skobe, Anita Rogic, Marcus Bosenberg. Characterization and role of CCR8+ regulatory T-cells in mouse models of malignant 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 B156.
Abstract B160: Comparative analysis of pharmacokinetics and antitumor effect between anti-PD-1 and anti-PD-L1 in mice models
Authors: Hatakeyama Hiroto , Kurino Taiki , Suzuki Hiroyuki , Terui Ayu , Uehara Tomoya , Arano Yasushi , Hisaka Akihiro
Published: February 2019

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.
Abstract B164: Inhibiting glycolysis enhances imiquimod-induced immunogenic cell death and the antitumor immune response
Authors: Hunag Shi-Wei , Shieh Jeng-Jer , Wang Sin-Ting , Cho Der-Yang , Chiu Shao-Chih
Published: February 2019

Immunogenic cell death (ICD) characterized as cells undergo with ICD could elicit a specific immune response to against a subsequent challenge with the same type living cells. Imiquimod (IMQ), a synthetic Toll-like receptor 7 ligand, is a imidazoquinoline family member and contains both antitumor and antiviral activity. IMQ is currently clinical used in topical, noninvasive treatment for various skin malignances. IMQ exerts its anti-tumoral activity through stimulating TLR7/8 in dendritic cells and activate cell-mediated immune responses, and directly induces tumor cell death. Recently, we had demonstrated IMQ directly induced autophagic cell death and apoptosis to against tumor cells that independent of TLR7/8. Thus, IMQ may induce tumor undergo ICD to trigger specific antitumor immune response. We found that IMQ induced expression of ICD-associated features in various cancer cell lines. We demonstrated that vaccination of IMQ-induced ICD cell lysate provided significant antitumor immunity with increased CD4 and CD8 T lymphocytes proliferation, tumor-specific cytotoxic lysis and infiltration of CD4, CD8, CD11c and IL-17A-producing cells in tumor lesion. Pharmacologic inhibition and genetic manipulation of ICD-associated features repressed IMQ-treated cell lysate-mediated specific anti-tumor immunity. We also found that IMQ-induced ICD-associated features is mediated by ROS production and ER stress through PERK/eIF2α pathway. In addition, inhibition of glycolysis significantly enhanced IMQ-induced ICD cell lysate-mediated antitumor immunity and ICD-associated features. Taken together, these results provided solid evidence to support that IMQ is an authentic ICD inducer and independent of TLR7/8 expression, and targeting aerobic glycolysis could be a desirable strategy to enhance IMQ-induced ICD and antitumor immunity.

Citation Format: Shi-Wei Hunag, Jeng-Jer Shieh, Sin-Ting Wang, Der-Yang Cho, Shao-Chih Chiu. Inhibiting glycolysis enhances imiquimod-induced immunogenic cell death and the antitumor immune response [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 B164.
Abstract B175: Semliki Forest virus-mediated oncolytic immunotherapy in mouse GL261 glioblastoma model
Authors: Martikainen Miika , Yu Di , Ramachandran Mohanraj , Fotaki Grammatiki , Martikainen Minttu-Maria , Merits Andres , Essand Magnus
Published: February 2019

Glioblastoma (GBM) is a devastating cancer of the central nervous system with no cure currently available. Oncolytic virotherapy with viruses that infect and destroy cancer cells provides a novel promising candidate therapy. We have previously shown that Semliki Forest virus (SFV) has therapeutic potency against orthotopic xenograft and syngeneic glioma models in mice. Here we show that SFV-infected cancer cells undergo immunogenic apoptosis, which triggers phagocytosis and maturation in co-cultured dendritic cells. SFV-killed GL261 mouse GBM cell lysate was also able to induce protective antitumor immune response in syngeneic mouse model, indicating that SFV oncolysis is immunogenic in vivo. By introducing mutations into the viral genome we have been able to produce a novel SFV clone which shows notably enhanced oncolytic potency in GL261 cells. As compared to the wild-type virus, the mutated SFV replicates faster and induces significantly stronger cytopathic effect. Robust viral replication and cytopathic effect were detected despite of activated type-I interferon signaling in the infected cells. This suggests that the enhanced SFV has increased resistance to antiviral response in mouse GBM cells. Viral proteins were detected by immunohistochemistry in orthotopic GL261 tumors after intratumoral injection of the mutated SFV virus. This provides evidence that the enhanced SFV is able to replicate in GL261 tumors in the face of antiviral in vivo microenvironment. Unwanted SFV replication in healthy brain cells can be inhibited by microRNA-mediated de-targeting. Taken together, the results indicate that oncolytic virotherapy with SFV can trigger antitumor immune responses, and support that SFV is a potent new candidate for oncolytic immunotherapy of GBM. These findings pave way for future clinical trials with oncolytic SFV.

Citation Format: Miika Martikainen, Di Yu, Mohanraj Ramachandran, Grammatiki Fotaki, Minttu-Maria Martikainen, Andres Merits, Magnus Essand. Semliki Forest virus-mediated oncolytic immunotherapy in mouse GL261 glioblastoma 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 B175.
Abstract B182: Repurposing antiviral T-cells to fight tumors
Authors: Rosato Pamela C. , Wijeyesinghe Sathi , Stolley J. Michael , Nelson Christine , Davis Rachel L. , Manlove Luke S. , Pennell Christopher A. , Blazar Bruce R. , Chen Clark C. , Geller Melissa A. , Vezys Vaiva , Masopust David
Published: February 2019

Overcoming the immunosuppressive tumor microenvironment and localizing adoptive cell and checkpoint blockade therapies to solid tumors remain major impediments to successful cancer immunotherapy. Humans experience many viral infections. Once controlled, the host retains memory T-cells throughout the entire body to sense reinfection or recrudescence. Mouse models have demonstrated that when that same virus is reencountered, these T-cells sound an alarm that induces a local immunostimulatory environment that activates and recruits many arms of the immune system. We observed that, like healthy tissue, human tumors are commonly surveyed by memory T-cells specific for previously encountered viral infections. Antiviral T-cell immune surveillance of tumors was recapitulated in mouse cancer models. We tested and discovered that local delivery of adjuvant-free peptide, derived from previously encountered mouse viral infections, recapitulated the sensing and alarm T-cell function within the tumor: recruiting and activating both the innate and adaptive immune system. This approach induced intratumoral accumulation of granzyme B+ CD8+ T and NK cells, and activated dendritic cells within the tumor and subsequently within the tumor draining lymph node. In addition to stimulating the tumor microenvironment, preliminary data suggest activated antiviral T-cells directly killed peptide coated tumor cells. Viral peptide administration arrested rapidly growing and poorly immunogenic B16 melanomas in vivo and this treatment synergized with anti-PD-L1 checkpoint blockade to eliminate measurable tumors, and prevented recurrence in 34% of mice. Most cured mice rejected subsequent B16 tumor challenges at distant sites, indicating that effective systemic tumor-specific immunity was established. In support of the hypothesis that this approach could translate to human cancer immunotherapy, we found that viral peptide alarm therapy of freshly isolated human tumors drove similar immune activation to that observed in mice. This study demonstrates that natural and existing antiviral immunity can be repurposed to fight tumors without the need for adjuvant, vaccination, or personalized identification of immunogenic tumor neoantigens.

Citation Format: Pamela C. Rosato, Sathi Wijeyesinghe, J. Michael Stolley, Christine Nelson, Rachel L. Davis, Luke S. Manlove, Christopher A. Pennell, Bruce R. Blazar, Clark C. Chen, Melissa A. Geller, Vaiva Vezys, David Masopust. Repurposing antiviral T-cells to fight tumors [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 B182.
Abstract B183: LFA-1 signals via Crk adapter proteins to induce actin-dependent T-cell migration and mechanosensing
Authors: Roy Nathan H. , MacKay Joanna L. , Buffone Alexander , Newell Krista , Robertson Tanner F. , Agarwal Sangya , Karimi Mobin , Hammer Daniel A , Burkhardt Janis K.
Published: February 2019

Allogeneic hematopoietic stem cell transplants (HSCT) are used to treat many malignancies, but the prevalence of graft-versus-host disease (GvHD) limits their overall success. Manipulating T-cell trafficking has emerged as an effective countermeasure, yet downstream integrin signaling pathways have yet to be targeted. We previously found that T-cells lacking the adapter proteins Crk and CrkL exhibit a robust antitumor response while causing little GvHD. We now find that T-cells from mice lacking Crk adapter proteins exhibit defects in LFA-1/ICAM-1 induced actin polymerization, leading edge formation, 2D migration, and integrin-mediated mechanosensing. Under shear flow, Crk/CrkL deficient T-cells fail to migrate upstream on ICAM-1 but migrate normally on VCAM-1, suggesting these integrin ligands relay different outside-in signals. Analysis of LFA-1 signaling reveals that Crk protein expression is required for phosphorylation of c-Cbl and its subsequent interaction with the PI3K subunit p85. Through this mechanism, Crk proteins promote PI3K activity and cytoskeletal remodeling downstream of LFA-1 engagement. Interestingly, this signaling pathway was largely specific to the LFA-1/ICAM-1 interaction, as WT-cells plated on VCAM-1 (which binds and signals through VLA-4) failed to induce high levels of phospho-Cbl, showed diminished PI3K activity, and did not migrate with a defined actin-rich leading edge. Finally, we show that knocking out CrkL alone is sufficient to alleviate GvHD, pointing toward unique roles of the Crk isoforms in T-cell biology. Together, these studies identify key signaling differences downstream of β2 vs β1 integrins that drive T-cell migratory behavior, and identify CrkL as an important factor during GvHD. Importantly, these data provide insight into integrin signaling that could be used to manipulate T-cell trafficking.

Citation Format: Nathan H. Roy, Joanna L. MacKay, Alexander Buffone Jr., Krista Newell, Tanner F. Robertson, Sangya Agarwal, Mobin Karimi, Daniel A Hammer, Janis K. Burkhardt. LFA-1 signals via Crk adapter proteins to induce actin-dependent T-cell migration and mechanosensing [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 B183.
Abstract B192: CD56 participation in immune effector cell activation and tumor cell eradication: A role for interleukin-15
Authors: Acker Heleen H. Van , Versteven Maarten , Reu Hans De , Ponsaerts Peter , Berneman Zwi N , Tendeloo Viggo F. Van , Smits Evelien L.
Published: February 2019

Oncoimmunologists are in a constant search for more effective immunotherapeutic treatments, helping to cure cancer. In this respect, immune cell participation is a key issue. A particularly interesting marker to identify antitumor immune cells is the neural cell adhesion molecule (NCAM), known as CD56. Namely, hematopoietic expression of CD56 seems to be confined to activated immune cells exhibiting some level of cytotoxic properties (Van Acker et al., Frontiers in Immunology 2017). Unfortunately, the current knowledge on the expression and functional role of CD56 is very fragmented. Therefore, we sought to elucidate the role of CD56 expression on various killer immune cells. First, we identified the high motility NCAM-120 isoform to be the main subset on immune cells. Next, through neutralization of surface CD56, we were able to demonstrate for the first time a direct involvement of CD56 in tumor cell lysis exerted by CD56-expressing killer cells such as natural killer cells, gamma delta (γδ) T-cells and interleukin (IL)-15-cultured dendritic cells (DCs). We also detected a putative crosstalk mechanism, suggesting CD56 as a co-stimulatory molecule in the interaction between IL-15 DCs and CD8 T-cells. Finally, by blocking the mitogen-activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase (PI3K)–AKT pathway, with respectively trametinib and afuresertib, we confirmed our hypothesis that IL-15 stimulation directly leads to CD56 upregulation via the recruitment of shc, binding a phosphotyrosine residue on the IL-2/15Rβ chain. In conclusion, these results underscore the previously neglected importance of CD56 expression on immune cells, benefiting current and future immune therapeutic options.

Citation Format: Heleen H. Van Acker, Maarten Versteven, Hans De Reu, Peter Ponsaerts, Zwi N Berneman, Viggo F. Van Tendeloo, Evelien L. Smits. CD56 participation in immune effector cell activation and tumor cell eradication: A role for interleukin-15 [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 B192.
Abstract PR08: Mechanisms of primary resistance to PD-1 checkpoint blockade
Authors: Krogsgaard Michelle , Moogk Duane , Li Kaitao , Yuan Zhou , Osman Iman , Weber Jeffrey S , Zhu Cheng
Published: February 2019

Although much clinical progress has been made in harnessing the immune system to recognize and target cancer, there is still a significant lack of an understanding of how tumors evade immune recognition and the mechanisms that drive tumor resistance to both T-cell and checkpoint blockade immunotherapy. Our objective is to understand how tumor-mediated signaling through inhibitory receptors, including PD-1, combines to affect the process of T-cell recognition of tumor antigen and activation signaling. This has the goal of understanding the basis of resistance to PD-1 blockade and potentially identifying new molecular targets to enable T-cells to overcome dysfunction mediated by multiple inhibitory receptors. Biomembrane Force Probe (BFP) measurements show that that the activities of TCR-proximal signaling components affect T-cell mechanosensing and sensitivity at the earliest stages of antigen recognition and are influenced by PD-1 and other inhibitory receptors via Shp-1/2 by targeting CD28 and Lck to directly suppress TCR-pMHC-CD8 binding. Phospho-proteomics and flow cytometry-based analysis of patient-derived T-cells from PD-1 responders and nonresponders identified additional mediators, signaling components and pathways associated with PD-1 checkpoint blockade resistance. Targeting these interactions and understanding the basis of resistance to PD-1 blockade would potentially allow identification of novel biomarkers of resistance or new molecular targets to enable T-cells to overcome dysfunction during PD-1 checkpoint blockade.

Citation Format: Michelle Krogsgaard, Duane Moogk, Kaitao Li, Zhou Yuan, Iman Osman, Jeffrey S Weber, Cheng Zhu. Mechanisms of primary resistance to PD-1 checkpoint blockade [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 PR08.
Abstract PR16: Mucosal-associated invariant T-cells respond to the cutaneous microbiota and promote skin immunity
Authors: Constantinides Michael G. , Tamoutounour Samira , Linehan Jonathan L. , Sen Shurjo , Shaik Jahangheer , Roy Sobhan , Adams Erin J. , Belkaid Yasmine
Published: February 2019

Infectious agents are estimated to cause approximately 18% of cancers worldwide, supporting a link between microbial products and carcinogenesis. The diverse array of commensal microorganisms that reside at barrier sites of the human body, collectively termed the microbiome, provide the primary source of constitutive microbial stimulation and promote immune homeostasis through their release of microbial products, including derivatives of vitamin synthesis. Mucosal-associated invariant T (MAIT) cells are unconventional T-cells that recognize microbial-derived metabolites and provide an initial defense to pathogens through their rapid production of type-1 or type-17 cytokines. While MAIT-cells have been shown to infiltrate colorectal tumors, it remains to be determined how MAIT-cells contribute to tumorigenesis and whether commensals regulate MAIT-cell function. Here we show that MAIT-cells are a substantial skin-resident population capable of producing IL-17A. IL-23 signaling promotes the accumulation of cutaneous MAIT-cells and skin commensals induce local MAIT-cell proliferation and IL-1-dependent IL-17A production. In the absence of commensals, MAIT-cells are dramatically decreased, indicating that the microbiota is necessary for the development of this population. Utilizing MAIT-cell-deficient mice, we demonstrate that cutaneous MAIT-cells promote wound healing and inhibit the colonization of pathogens. This work identifies MAIT-cells as the only cell population that is entirely dependent on the microbiota and reveals the mechanism by which these cells respond to the commensal microbial community. Since IL-17A promotes tumor vascularization and neutrophil recruitment, the high frequency of MAIT-cells in human skin suggests that modulation of the skin-resident bacteria may have clinical applications for the progression of melanoma, and current studies are exploring this possibility using the B16 melanoma model in mice.This work was supported by the NIH Intramural Research Program. MGC is a Cancer Research Institute Irvington Fellow supported by the Cancer Research Institute.

Citation Format: Michael G. Constantinides, Samira Tamoutounour, Jonathan L. Linehan, Shurjo Sen, Jahangheer Shaik, Sobhan Roy, Erin J. Adams, Yasmine Belkaid. Mucosal-associated invariant T-cells respond to the cutaneous microbiota and promote skin immunity [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 PR16.
Abstract A21: Activation of 4-1BB on liver myeloid cells triggers hepatitis via an interleukin-27 dependent pathway
Authors: Jaiswal Ashvin R. , Bartkowiak Todd , Ager Casey R. , Chin Renee , Chen Chao Hsien , Budhani Pratha , Ai Midan , Reilley Matthew J. , Sebastian Manu M. , Hong David , Curran Michael A.
Published: February 2019

Purpose: Agonist antibodies targeting the T cell co-stimulatory receptor 4-1BB (CD137) are among the most effective immunotherapeutic agents across multiple preclinical models of cancer. In the clinic, however, development of these agents has been stymied by dose-limiting liver toxicity. Lack of knowledge of the mechanisms underlying this toxicity has limited the potential to separate 4-1BB agonist driven anti-tumor immunity from hepatotoxicity.

Experimental Design: The capacity of 4-1BB agonist antibodies to induce liver toxicity was investigated in immune competent mice, with or without co-administration of checkpoint blockade, via measurement of serum transaminase levels, through imaging of liver immune infiltrates, and via qualitative and quantitative assessment of liver myeloid and T cells via flow cytometry. Knockout mice were used to clarify the contribution of specific cell subsets, cytokines and chemokines.

Results: We find that activation of 4-1BB on liver myeloid cells is essential to initiate hepatitis. Once activated, these cells produce interleukin-27, which is required for liver toxicity. CD8 T cells infiltrate the liver in response to this myeloid activation and mediate tissue damage triggering transaminase elevation. FoxP3+ regulatory T cells limit liver damage and their removal dramatically exacerbates 4-1BB agonist hepatitis. Co-administration of CTLA-4 blockade ameliorates transaminase elevation, whereas PD-1 blockade exacerbates it. Loss of the chemokine receptor CCR2 blocks 4-1BB agonist hepatitis without diminishing tumor-specific immunity against B16 melanoma.

Conclusions: 4-1BB agonist antibodies trigger hepatitis via activation of myeloid cells to produce Interleukin-27. Co-administration of CTLA-4 and/or CCR2 blockade may minimize hepatitis but yields equal or greater antitumor immunity.

Citation Format: Ashvin R. Jaiswal, Todd Bartkowiak, Casey R. Ager, Renee Chin, Chao Hsien Chen, Pratha Budhani, Midan Ai, Matthew J. Reilley, Manu M. Sebastian, David Hong, Michael A. Curran. Activation of 4-1BB on liver myeloid cells triggers hepatitis via an interleukin-27 dependent pathway [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A21.
Abstract A31: Reprogramming of tumor-associated macrophages by anti-Program Death Ligand 1 (PDL1)
Authors: Xiong Huizhong , Moskalenko Marina , Rodriguez Ryan , Mittman Stephanie , Yang Michelle , Kim Jeong , Barcelli Rafael Cubas
Published: February 2019

Tumor-associated macrophages play a vital role in shaping tumor environment and contribute to the cancer-immune set point. Immunotherapy such as aPD1/aPDL1 has demonstrated remarkable therapeutic efficacy on a variety of cancers through reinvigorating CD8+ T cells. However, its impact on macrophages largely remains unclear. Here by using mouse MC38 cancer model, we show that aPDL1 re-programs macrophages from M2-like to M1-like: it decreases Arginase-I (ARG1), an M2-like marker, and increases iNOS and MHCII, both M1-like markers, on macrophages. Next, we investigate the mechanism behind the reprogramming. We identify that IFNg from CD8+ T cells and NK cells is boosted by aPDL1 and is required for macrophage re-polarization. IFNg blockade abrogates this process. Finally, we examine additional cancer models and observe similar phenotypes in another aPDL1-responsive model, but not in a third, non-responsive model. We conclude that tumor associated macrophage is re-programmed from M2-like to M1-like following aPDL1 treatment, and that it is mediated by enhanced IFNg in the tumor environment. Our findings suggest macrophage reprogramming as a novel mechanism and an independent avenue to promote antitumor activity via modulation of the cancer-immune set point.

Citation Format: Huizhong Xiong, Marina Moskalenko, Ryan Rodriguez, Stephanie Mittman, Michelle Yang, Jeong Kim, Rafael Cubas Barcelli. Reprogramming of tumor-associated macrophages by anti-Program Death Ligand 1 (PDL1) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A31.
Abstract A40: Prevalence of TIGIT expression in normal tissues, inflammation, and cancer
Authors: Hinsch Andrea , Kluth Martina , Lübke Andreas M. , Menz Anne , Krech Till , Steurer Stefan , Höflmayer Doris , Sauter Guido , Wilczak Waldemar , Fischer Kristine , Simon Ronald
Published: February 2019

Introduction: TIGIT (T-cell immunoreceptor with Ig and ITIM domains) is an immune checkpoint protein expressed on subsets of cytotoxic and regulatory T cells. TIGIT inhibiting drugs are currently being developed. The purpose of this study was to investigate the pattern of TIGIT expressing cells in normal tissues, inflammation and cancer.

Methods: Monoclonal mouse antibodies were used for immunohistochemical TIGIT (Dianova, Hamburg, Germany) and PD1 (Abcam, Cambridge, UK, ab52587) analyses on formalin fixed paraffin embedded tissue sections from normal lymphatic tissues as well as selected inflammations and cancers.

Results: In lymph nodes and tonsils, TIGIT positivity was seen in a large fraction of the germinal center T-cells. A high density of TIGIT positive T-cells also occurred in the marginal zone and in the interfollicular area. The analyses of selected inflammatory diseases revealed detectable TIGIT expression in a high proportion of T-lymphocytes in BCG induced cystitis and prostatitis, Hashimoto thyreoiditis, Lichen sclerosis of the penis, skin eczema, Crohn’s disease, and rheumatoid synovialitis. In all normal and inflammatory tissues, the pattern of TIGIT expression largely paralleled the pattern of PD1 expression. Three examples each of cancers types for which immune checkpoint inhibitors are already approved or extensively studied (melanoma, lung, kidney, bladder and colorectal cancer) were also analyzed. Here, the number of CD8 positive lymphocytes varied greatly between individual cases and the fraction of TIGIT and PD1 positive CD4 and CD8 positive lymphocytes varied considerably between cases.

Conclusion: Expression of TIGIT is regularly seen in a large subset of T-cells and has a similar expression pattern as PD1. TIGIT’s frequent co-expression with PD1 in cytotoxic T-cells is consistent with TIGIT representing a clinically relevant druggable immune checkpoint regulator that potentially could be targeted in combination with PD1.

Citation Format: Andrea Hinsch, Martina Kluth, Andreas M. Lübke, Anne Menz, Till Krech, Stefan Steurer, Doris Höflmayer, Guido Sauter, Waldemar Wilczak, Kristine Fischer, Ronald Simon. Prevalence of TIGIT expression in normal tissues, inflammation, and cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A40.
Abstract A42: CD8+PD-1+ cells population were associated with the superiority of DC/CIK cell immunotherapy combined S-1 in patients with advanced pancreatic and gastric cancer
Authors: Ren Jun , Qiao Guoliang , Wang Xiaoli , Zhou Xinna , Huang Lefu
Published: February 2019

Purpose: Advanced pancreatic(APC) and gastric cancer(AGC) remain challenging to treat effectively. This study aims to investigate the potential role of CD8+PD-1+ T-cells population on the superiority of immunotherapy with dendritic cells and cytokine induced killer cells (DC-CIK) administered with the chemotherapy(S-1).

Methods: Consecutive patients with advanced pancreatic cancer (n=36) and gastric cancer (n=30) were treated with either DC-CIK or DC-CIK+S-1. CIK cells were expanded with IL-2. Flow cytometry and ELISPOT were used for sorting and testing the reactivity CD8+PD-1+ T-cells. Survival estimates were calculated according to the Kaplan and Meier methodology.

Results: The median overall survival(OS) and progression free survival(PFS) for DC-CIK plus S-1 were 212 and 136 days which were significantly higher than other groups for treatment of APC(P<0.001). Similarly, significant differences in PFS and OS were showed in DC-CIK plus S-1 for treatment of AGC(P<0.001). PD-1- and PD-1+ DC-CIK cells were cocultured with their autologous tumor cell lines, and reactivity was assessed by measuring IFN-γ secretion. We found a clear correlation between the percentage of CD8+PD-1+ T-cells on day 7 of culture and the reactivity of the DC-CIKs after 14 days in culture with IL-2. Moreover, CD8+PD-1+ T-cells showed higher tumor specific IFN-γrelease in ELISPOT, compared to CD8+ PD-1- sorted T-cells or unsorted. Further survival analysis showed that patients with the percentage of CD8+PD-1+ T-cells population enhanced more than 2 times after culture had favorable OS and PFS compared with those having no CD8+PD-1+ T-cells increasing. These results showed that CD8+PD-1+ T-cells population might be an excellent biomarker for tumor or antigen-specific activation of adoptive cell therapy.

Conclusion: CD8+PD-1+ T-cells populations were associated with the superiority of DC/CIK combined S-1 and could be a useful biomarker for enriching tumor specific T-cells for cell immunotherapy of patients with APC and AGC.

Citation Format: Jun Ren, Guoliang Qiao, Xiaoli Wang, Xinna Zhou, Lefu Huang. CD8+PD-1+ cells population were associated with the superiority of DC/CIK cell immunotherapy combined S-1 in patients with advanced pancreatic and gastric cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A42.
Abstract A62: NKG2D RNA CAR is effective in treating peritoneal carcinomatosis in a mouse model
Authors: Li Zhendong , Zha Shijun , Wang Shu
Published: February 2019

Peritoneal carcinomatosis (PC) is widely shown in end-stage neoplastic disease, especially in recurrent colorectal cancer, and has become a significant threat to the overall survival of patients. Since NKG2D ligands are commonly up-regulated on cell surface of carcinomas but rarely present on most healthy tissues, they can be the ideal targets in the treatment of NKG2D ligand-overexpressing colorectal PC. In this study, we developed a chimeric antigen receptor (CAR)-T cell therapy approach by arming human T lymphocytes with mRNA coding a chimeric NKG2D receptor to target NKG2D ligands. We constructed first, second, and third generation CARs specific for NKG2D ligands by linking the extracellular domain of the human NKG2D receptor to the CD3-zeta signaling domain and costimulatory moieties. mRNAs of those chimeric NKG2D CARs were electroporated into human T lymphocytes and the anti-tumor activities of engineered T cells were examined afterwards. The NKG2D CAR expression was detectable for at least 6 days and the modified T cells exhibited efficient cytotoxic immune activity against NKG2D ligand-positive tumor cells, but not NKG2D ligand-negative cells. Multiple infusions of the RNA CAR modified T cells in immunodeficient mice bearing established peritoneal human colorectal tumors led to a significant reduction in the tumor burden. Here, we showed that T cells engineered with NKG2D CAR by RNA electroporation can efficiently control the colorectal peritoneal carcinomatosis in the mouse model. Transfection of mRNA encoding a CAR is an economical way to benefit the test of new CARs and it holds great promise in controlling on-target/off-tumor toxicity and cytokine storms. The possibility of using NKG2D ligand-specific RNA CARs to treat colorectal PC warrants further investigation.

Citation Format: Zhendong Li, Shijun Zha, Shu Wang. NKG2D RNA CAR is effective in treating peritoneal carcinomatosis in a mouse model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A62.
Abstract A66: Analysis of dendritic cell derived exosomes that suppressed tumor growth
Authors: Takanashi Masakatsu , Sudo Katsuko , Ueda Shinobu , Ohno Shinichiro , Kuroda Masahiko
Published: February 2019

Immunotherapy is expected as the fourth cancer therapy subsequently to the three pillars of cancer treatment which are surgery, radiation, and chemotherapy. Cancer immunotherapy enhances existing antitumor responses and includes the use of antibodies, lymphocytes and cytokines. Dendritic cells (DCs)-based immunotherapy is one of the cancer immunotherapies. Because DCs play a key role for immune reactions to activate T cells against cancer cells by cancer antigen presentation at cellular membrane, DCs have been used in clinical trials as cellular mediators for therapeutic vaccination of patients with cancer. The cells such as DCs and cancer cells secrete exosomes which are nano-sized extracellular microvesicles. It has reported that the exosomes released from peptide-vaccinated DCs are responsible for the persistence of antigen presentation. On the other hands, cancer cells derived exosomes play an immunosuppressive. So, we considered that whether DCs-derived exosomes could induce suppress cancer cells and more effective response of immune system against cancer with control for the cancer cells-derived exosomes. Luciferase gene transfferd-3LL cells (murine lung cancer cell line derived C57BL/6) were injected to C57BL/6J mice by intraperitoneal administration. And then, DCs, DCs-exosomes or 3LL-exosmes were weekly administrated to lung cancer bearing mice. The exosomes derived from DCs decreased lung cancer cell growth, on the other hand, lung cancer derived-exosomes increased in compared with DCs, DCs-exosomes and non-treated. For cancer immunotherapy, DC-exosomes and controlled cancer-exosomes play important roles. Currently, we are going on analyze immunosuppressive molecules possessing cancer cell-derived exosomes, and immune activation molecules in DCs-exosomes.

Citation Format: Masakatsu Takanashi, Katsuko Sudo, Shinobu Ueda, Shinichiro Ohno, Masahiko Kuroda. Analysis of dendritic cell derived exosomes that suppressed tumor growth [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A66.
Abstract A67: Bi-phasic metabolic responses to in situ macrophage activation
Authors: Kam Yoonseok , Swain Pamela M. , Dranka Brian P.
Published: February 2019

The cellular metabolism of macrophages is an emerging element regulating inflammatory macrophages which are a critical component of tumor microenvironment. The inflammatory macrophage with highly glycolytic phenotype is also known to elevate the glycolytic activity upon pathogenic stimulation such as lipopolysaccharide (LPS). In this study, the dynamic changes in glycolysis were traced in a real-time manner by measuring proton efflux rates (PERs) and oxygen consumption rates (OCR) after an in-situ activation using Seahorse XFe96 analyzer. The PER of human peripheral blood monocyte (PBMC) derived M1 macrophages was increased within an hour after injection of LPS, which corresponding to cytokine release, tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β). In contrast to PBMC-derived M1 macrophage activation, macrophage cell lines of RAW264.7 and J774.A1 required co-stimulation with interferon γ (IFNγ) for the full activation. Interestingly, the LPS and IFNγ co-stimulation modulates glycolytic rates in a bi-phasic manner which was identified only in long term (> 6 hr) monitoring. A series of long term XF analysis using in situ activation revealed that the immediate early glycolytic response fully relies on LPS stimulation while the secondary elevation in PER depends on IFNγ stimulus, which turns on inducible nitric oxide synthase (iNOS) signaling and in turn suppresses mitochondrial respiration. The TNFα production is closely related to the immediate early glycolytic elevation, but independent from IFNγ-induced second elevation. The IFNγ-dependent second glycolysis increase was totally abolished by iNOS inhibitors whereas the immediate early glycolysis elevation was not affected at all. These data imply a temporal orchestration mechanism of LPS and IFNγ signaling in the metabolic regulation and activation of inflammatory macrophages.

Citation Format: Yoonseok Kam, Pamela M. Swain, Brian P. Dranka. Bi-phasic metabolic responses to in situ macrophage activation [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A67.