Trends Immunol Research Articles

A phase 1/1b, first-in-human, multi-part study of DF6215, an engineered IL-2Rα-active agonist, to investigate the safety, tolerability, pharmacokinetics, and biological and clinical activity in patients with advanced solid tumors.

TPS2704 Background: IL-2 is a pleiotropic cytokine that stimulates strong anti-tumor responses by promoting expansion and activation of cytotoxic tumor-infiltrating immune cells including CD8 + T cells and NK cells (1). Aldesleukin is an approved rhIL-2; however, it has clinical limitations due to short half-life, requirement for 5 days of hospitalization per cycle, and high toxicity [eg, cytokine-related toxicity, capillary leak syndrome (CLS)] that limits its use [aldesleukin USPI]. DF6215 is a modified monovalent human IL-2 that binds to the IL2R. Preclinically, DF6215 administration results in sustained and modulated IL-2 pharmacology, leading to protracted pharmacodynamic (PD) effects and enhanced anti-tumor activity without observation of CLS in murine and primate toxicology studies. In response to the limitations of previous IL-2 therapeutics, DF6215 was designed as an IL-2Rα-active agonist with increased IL-2Rβγ stimulation to improve the benefit-to-risk ratio of historic IL-2 drugs. An additional design element is a prolonged elimination half-life due to Fc fusion, allowing for less frequent dosing. Methods: This is a first-in-human, open-label, multipart, Phase 1/1b clinical trial to characterize the initial safety, tolerability, pharmacokinetics, and preliminary efficacy of DF6215 in patients with advanced solid tumors. The Phase 1 dose escalation (DE) starting dose is 10 ug/kg IV every 2 weeks (up to 42 subjects expected to be enrolled), and the Safety/PK/PD will be explored at doses already characterized as safe in dose escalation and potentially efficacious (max of 40 subjects). Phase 1b includes an efficacy expansion (EE) cohort in subjects with advanced melanoma who have received prior anti-PD-1 agents and, if the tumor harbors a BRAF-activating mutation, a BRAF inhibitor. Key inclusion criteria include ≥ 18 years of age; ECOG PS of 0 or 1; adequate organ function, and locally advanced or metastatic solid tumors for which standard therapy options have been exhausted; and availability of baseline tumor biopsy. Primary objectives include determination of the MTD of DF6215 in DE and demonstration of clinical activity of DF6215 in the EE. Secondary objectives include assessment of safety and tolerability, pharmacokinetics, and immunogenicity of DF6215. This study plans to enroll up to 102 subjects across ~30 global centers in the US, European, and the Asia/Pacific regions, and is currently open to enrollment. 1. Arenas-Ramirez N, et al. 2015. Trends Immunol. Clinical trial information: NCT06108479 .

Classification of NK-Large Granular Lymphocytic Leukemia By CD56 Expression

Introduction Large granular lymphocytic leukemia (LGLL) is a rare chronic lymphoproliferative disease of T cell and natural killer (NK) cell lineage. Less than 10% of cases are NK-large granular lymphocytic leukemia (NK-LGLL), which is distinguished by clonal growth of mature NK cells that are CD3-, CD16+, and/or CD56+. There are two types of mature NK cells based on CD56 intensity. The CD56dim NK-cell subset exhibits a higher level of natural cytotoxic activity compared to the CD56bright NK-cell subset, which, in turn, is capable of producing abundant cytokines ( Trends Immunol. 2001;22(11):633-640). However, few studies have explored the internal heterogeneity of NK-LGLL based on CD56 expression ( Leukemia. 2010;24(4):881-884; Blood Cancer J. 2018;8(6):51) . Method A total of 402 patients have been diagnosed with LGLL at our hospital since June 2005, including 40 with NK-LGLL. All patients met the recommended diagnostic criteria ( Blood. 2017;129(9):1082-1094). Treatment responses were collected after at least 3 months of therapy. NK subtype was defined by flow cytometry analysis using common surface markers showing CD3−/CD16+, or CD56+ pattern, and cells were considered antigen partial positive if antibody staining was between 20%-80%. To perform subgroup analysis, we incorporated partial CD56 positive into the negative group since mature NK cells are CD56-positive. Clonality was evaluated by killer-cell immunoglobulin-like receptors (KIR). Sequencing of STAT3 and STAT5b was performed with Next Generation Sequencing. Results Twenty-eight of 40 patients (70.0%) presented symptoms at the time of diagnosis in which anemia was the most prevalent accounting for 67.5%. PRCA was the most common comorbidity, affecting 20.0% of patients. All cases were CD2+, CD3-. Expressions of mature NK cell markers are heterogenous, including CD8 (22.5%), CD16 (77.5%), CD56 (40.0%), CD57 (58.9%), CD94 (78.8%), CD161 (35.3%), perforin (83.3%), GramB (77.8%). KIR analyses were available in 31 cases, 45.2% of NK-LGLs showed a form of restrictive expression, mainly CD158i (32.3%), an activating KIR, whereas 58.1% of cases lacked CD158a/b/e/f/i expression. STAT3 mutated status was studied in 28 cases (70.0%), 46.4% had STAT3 mutations, and no STAT5B mutations were detected. We classified 40 cases into two subsets based on CD56 expression (Table 1). CD56- cases displayed a more aggressive course. They usually presented with symptomatic disease including anemia (91.7% vs 31.3%, P= 0.000), severe anemia (66.7% vs 12.5%, P= 0.001), neutropenia (66.7% vs 12.5%, P= 0.001), splenomegaly (52.4% vs 12.5%, P= 0.012), and treatment requirement (87.0% vs 31.3%, P= 0.001). CD56-negative cases expressed CD161 at a higher frequency (60.0% vs 0.0%, P=0.001) while the other mature markers were similar. Interestingly, STAT3 mutation was detected in 13 of 20 (65.0%) in CD56- NK-LGLL, and none of 8 CD56+ NK-LGLL had STAT3 mutation (p=0.007). The treatment pattern was similar between CD56+ vs CD56- NK-LGLL. However, the treatment efficacy was lower in CD56- NK-LGLL, with overall response rates (ORR) of 100% vs 79% and CR (complete response) rate of 80% vs 46%, in CD56+ and CD56- NK-LGLL patients respectively. Conclusions NK-LGLL is a heterogenous entity and can be divided into two groups further based on CD56 expression. CD56- NK-LGLL showed more prominent clinical symptoms, high STAT3 mutation, and CD161 expression. CD56- NK-LGLL showed a poor response than CD56+ NK-LGLL. More investigations are deserved to explore the underlying mechanisms.

Circulating Mitochondrial DNA as a Diagnostic Biomarker for Predicting Disease Severity in Patients With Acute Pancreatitis

Acute pancreatitis (AP) is a leading cause of gastrointestinal hospital admissions with up to 40% mortality in moderate to severe AP. 1 Boxhoorn L. et al. Lancet. 2020; 396: 726-734 Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar Cell death and inflammation are key processes in the pathophysiology of AP, and mitochondria-mediated cell death plays a central role in the initiation of inflammatory pathways of AP. 2 Krysko D.V. et al. Trends Immunol. 2011; 32: 157-164 Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar Evidence suggests that several damage-associated molecular patterns (DAMPs) are released from dead pancreatic acinar mitochondria into the extracellular circulation. Given the need for early diagnosis of AP severity for proper treatment, we investigated whether circulating levels of mitochondrial DNA (mtDNA) fragments are a potential biomarker of early distinction for AP severity.

Immunomodulatory Functions of Glycolipids from Pathogens.

The cell envelopes of pathogens comprise a wealth of unique glycolipids, which are important modulators of the host immune responses during infection and in some cases have been used as adjuvants. Despite this abundant basic knowledge, the identities of the host immune receptors for mycobacterial lipids have long been elusive (Ishikawa et al., Trends Immunol 38:66-76, 2017). We describe the method of how to isolate glycolipids from microorganisms and how to analyze the glycolipids' potential to activate reporter cells and bone marrow-derived dendritic cells (BMDCs), such as surface marker expression and reactive oxygen species (ROS) production. Additionally, we outline an in vitro BMDC/T cell coculture model to investigate functional consequences of leukocyte activation, such as cytokine production. In this chapter, we provide a guide for extracting glycolipids from microorganisms and how to use them to activate leukocytes. We also present methods on how to generate and activate reporter cells, as well as BMDCs and how to set up BMDC/T cell cocultures. We further outline how to generate samples and how to analyze the immunomodulatory effect glycolipid exposure has on these cells, via flow cytometry, ROS production assays and ELISA.

39 A multi-modal analysis approach leveraging multiplexed spatial phenotyping and multi-omics analysis to better understand the prognostic value of tertiary lymphoid structures in NSCLC

BackgroundTertiary Lymphoid Structures (TLS) are highly organized ectopic lymphoid structures found in inflamed or tumor tissues, acting as sites of lymphoid recruitment and immune activation. A high TLS density within the tumor is commonly associated with an increased prognostic effect of TILs and with an improved disease free survival and overall survival for patients.1 However, the existence of conflicting studies suggest that multiple TLS features should be taken into account when assessing their prognostic value, such as their location, cellular composition, maturation stage and spatial organisation, as those may affect their functionalities.2MethodsWith the aim of gaining insights into TLS biology and evaluating the prognostic role of TLS in Non-Small Cell Lung Carcinoma according to their multiple features, we developed a TLS multiplex immunofluorescent (mIF) panel that includes T cells (CD3, CD8), B cells (CD20), Follicular Dendritic cells (CD21, CD23) and mature dendritic cells (DC-LAMP) markers. We deployed this panel across a cohort of primary tumors from NSCLC patients (n=408) and established a mIF image analysis workstream to assess the status and spatial location of each cell within the tissue. A H&E staining of the same tissue section was performed to evaluate mIF spatial data in relation to the tumor context. Additional multi-omics assessments were conducted across the same cohort including; whole exome sequencing, NanoString transcriptomics, and immunohistochemistry (e.g. PD-L1, FOXP3, NKp46, LKB1, CTLA4). We have leveraged clinical metadata, including demographics (e.g. age, sex, smoking status) and clinical risk factors (e.g. stage, grade, Standard of Care treatment) with clinical follow up (e.g. OS, PFS) for prevalence analysis, novel biomarker identification, and survival association.ResultsAssessment of the prevalence of each cell phenotype within the tumor tissue and TLS, the cell-cell interactions, the distance between each cell type, and the distance of non-TLS immune cells to the closest TLS will be described, demonstrating the different types of lymphoid aggregates and TLS and their functional status. An integrative analysis combining spatial biology data with multi-omics and clinical data will be presented evaluating the prognostic value of TLS composition, maturation status and spatial organization, in correlation with additional biomarkers and clinical characteristics.ConclusionsThis exploratory study using cutting-edge technologies enables us to better understand how TLS orchestrate an organised anti-tumour response, defining TLS spatial biomarker signatures, TLS gene signatures, and TLS features associated with patient outcomes to evaluate in the clinic.ReferencesMarie-Caroline Dieu-Nosjean, Jérémy Goc, Nicolas A Giraldo, Catherine Sautès-Fridman, Wolf Herman Fridman. Tertiary lymphoid structures in cancer and beyond. Trends Immunol 2014;35(11):571–580.Catherine Sautès-Fridman, Florent Petitprez, Julien Calderaro, Wolf Herman Fridman. Tertiary lymphoid structures in the era of cancer immunotherapy. Nat Rev Cancer 2019;19(6):307–325.Ethics ApprovalThe study was approved by AstraZeneca.

Steering a Course through the COVID-19 Pandemic: Should the SECURE-IBD Registry Influence Prescribing for Patients with Inflammatory Bowel Disease?

Summary The global pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, also referred to as COVID-19) rapidly became an unprecedented healthcare challenge. It represents a particular concern for patients with inflammatory bowel disease (IBD), many of whom require treatment with immunosuppressive medication which may be associated with an increased risk of both viral and bacterial infections (Gastroenterology 2018;155(2):337-346.e10). Moreover, as IBD is a lifelong condition, patients are frequently older with comorbidities, further increasing the potential risks of a more severe COVID-19 disease course. This has led to considerable uncertainty over appropriate treatment of IBD in the COVID-19 era, and has brought safety considerations into the focus of the patients and physicians.

SAT-026 Association of Inflammatory Markers with Depressive Symptoms Across the Perinatal Period

Perinatal depression (PND) is a mood disorder affecting 10-15% of women during pregnancy and postpartum. Its aetiology is complex with contribution from both genetic background and psychosocial as well as environmental stressors that determine individual responses shaped by chronic and acute disease burden (1). It is thought that the molecular basis of PND involves dysregulation of the HPA axis associated with neurotransmitter and neuroactive steroids imbalance. Inflammation appears to be a contributing mechanism, with increased levels of cytokines exerting adverse effects on serotonin metabolism, neuroplasticity and HPA hyperactivity (2). With only 50% of women detected through current screening strategies, there is an urgent unmet need for the development of biomarker-based strategies to identify women at risk of PND. In this study we used data and blood samples from the prospective Coventry and Warwickshire PND study; we investigated for inflammatory markers associated with depressive symptoms, assessed using the Edinburgh Postnatal Depression Score (EPDS) questionnaire between 24-29 weeks of gestation and again 6-10 weeks postpartum. A cut-off score of 10 categorize ‘high’ or ‘low’ risk for depression. Blood samples collected at 28 weeks of gestation were profiled for either IL-6 and IL-10 levels or a panel of 92 inflammatory markers. Individual inflammatory markers were compared across groups using Welch’s ANOVA. Results suggest that IL-10 levels were significantly correlated with EPDS score, exerting a protective effect (r= -.10), with reduced levels in the highest severity category (EPDS ≥ 15). The IL-6/IL-10 ratio was also associated with a raised EPDS score (r=.10, p=.01), as well as delivery complications (r=.09). The highest IL-6/IL-10 ratio is observed in women who had emergency caesarean section. Bayes’ theorem analysis suggested that IL-6/IL-10 ratio could be used as a negative screen to rule out low risk pregnancies. From the 92 inflammatory markers, 14 analytes were below the limit of detection for more than 50% of samples and so were excluded from further analysis. Upon comparison of groups determined by antenatal and postnatal EPDS scores, 29 markers displayed a significance value of P<0.05. Upon the application of post hoc tests, 8 markers including: STAM-BP, SIRT2, CD40, CASP8 and ADA, all associated with apoptotic processes, remained statistically significant in pregnant women with raised antenatal EPDS scores. This data support an association between inflammatory markers and perinatal depression and adverse pregnancy outcomes. Detailed quantitative analysis of such biomarker signatures at different stages of pregnancy, might lead to early detection of disease and application of targeted treatment. (1) Pariante, C. M. & Lightman, S. L. (2008) Trends Neurosci, 31 (9): 464-468. (2) Raison et al., (2006) Trends Immunol, 27 (1): 24-31.

Oral Candidiasis in a Migraine Patient Taking Erenumab and Galcanezumab: a Case Report

Currently, there are three FDA-approved injectable calcitonin gene-related peptide (CGRP) antagonists, namely erenumab-aooe, galcanezumab-gnlm, and fremanezumab, all approved in 2018. There is no documented evidence of any fungal infections with these medications. We report a patient with oral candidiasis after administration of erenumab and galcanezumab for migraine headaches. Per the literature, the presence of candida stimulates sensory neural tissue to release CGRP, which then in turn leads to the signaling of dendritic cells to release vital cytokines such as interleukin-23 (IL-23) (Kashem et al. in Trends Immunol. 37(7):440–50, 2016). CGRP is involved in the cutaneous exposure to antigens, leading to its release and subsequent signaling pathway. The disruption of this cascade could lead to a decrease in the innate immune response to candida infection, thus leading to its proliferation. A 48-year-old female with a diagnosis of migraine headache presents with oral candidiasis after anti-CGRP medications, erenumab, and galcanezumab, an adverse effect not yet reported to our knowledge. Long-term anti-CGRP effects on human physiology are not yet understood due to the relatively novelty of these medications. We review the literature to support our proposition that CGRP is involved in antifungal immunity, and its antagonism incurs susceptibility to candidiasis.

Pancreatic Tumor Microenvironment.

The pancreatic ductal adenocarcinoma (PDAC) microenvironment is a diverse and complex milieu of immune, stromal, and tumor cells and is characterized by a dense stroma, which mediates the interaction between the tumor and the immune system within the tumor microenvironment (TME). The interaction between stromal and tumor cells signals and shapes the immune infiltration of TME. The desmoplastic compartment contains infiltrated immune cells including tumor-associated macrophages (TAMs) and large numbers of fibroblasts/myofibroblasts dominated by pancreatic stellate cells (PSCs) which contribute to fibrosis. The highly fibrotic stroma with its extensive infiltration of immunosuppressive cells forms the major component of the pro-tumorigenic microenvironment (Laklai et al. Nat Med 22:497-505, 2016, Zhu et al. Cancer Res 74:5057-5069, 2014) provides a barrier to the delivery of cytotoxic agents and limits T-cell access to tumor cells (Feig et al. Proc Natl Acad Sci USA 110:20212-20217, 2013, Provenzano et al Cancer Cell 21:418-429, 2012). Activated PSCs reduced infiltration of cytotoxic T cells to the juxtatumoral stroma (immediately adjacent to the tumor epithelial cells) of PDAC (Ene-Obong et al. Gastroenterology 145:1121-1132, 2013). M1 macrophages activate an immune response against tumor, but M2 macrophages are involved in immunosuppression promoting tumor progression (Noy and Pollard Immunity 41:49-61, 2014, Ruffell et al. Trends Immunol 33:119-126, 2012). The desmoplastic stroma is reported to protect tumor cells against chemotherapies, promoting their proliferation and migration. However, experimental depletion of the desmoplastic stroma has led to more aggressive cancers in animal studies (Nielsen et al. World J Gastroenterol 22:2678-2700, 2016). Hence reprogramming rather than simple depletion of the PDAC stroma has the potential for developing new therapeutic strategies for PC treatment. Modulation of PSCs/fibrosis and immune infiltration/inflammation composes the major aspects of TME reprogramming.

AN UNUSUAL INFLAMMATORY RESPONSE IN TB LYMPHADENITIS IN ACUTE MYELOID LEUKEMIA PATIENTS

SESSION TITLE: Global Case Report Posters SESSION TYPE: Global Case Reports PRESENTED ON: 10/23/2019 09:45 AM - 10:45 AM INTRODUCTION: Tuberculosis has been the most common of the opportunistic infection in immunocompromised patients. In 2014, 9.6 million people suffered from tuberculosis and 1.5 million died from the disease. Among hematologic malignancies, the prevalence of tuberculosis is highest in acute myeloid leukemia (AML), ranging between 3.1-6.9%. CASE PRESENTATION: We report 2 cases of acute monoblastic leukemia patients who were simultaneously diagnosed with tuberculosis.They presented with febrile neutropenia after chemotherapy. Chest and abdominal CT revealed multiple intrathoracic lymphadenopathies and hepatosplenic microabscess in both cases. Microbiological diagnosis from intrathoracic lymph nodes obtained by EBUS-TBNA confirmed acid fast bacilli and culture for tuberculosis subsequently reported Mycobacterium tuberculosis in both cases. Although the patients suffered from neutropenia, the pathology of the intrathoracic lymph nodes revealed neutrophil as the cell response to tuberculosis. Remarkable clinical improvement, including fever, was accomplished following antituberculous medications. DISCUSSION: Previous studies reported the prevalence of concomitant TB and AML was 3.1-6.9%, highest among hematologic malignancies. The reason for higher prevalence of TB in AML patients is not clear and could be result of monocyte/macrophage dysfunction. In addition, function of T lymphocytes and NK cells in AML patients is impaired. Myeloblasts produce factors, which suppress T cell activation and proliferation causing immunity impairment. The studies in the role of neutrophils in host defense mechanism against TB investigated were limited and the results remain controversy. Influx of neutrophils associates with tissue destruction leading to secondary necrosis, which induces further inflammation. In the context of acquired immune response impairment, as in our cases, neutrophils play a role as a compensation for inadequate functioning of mononuclear phagocytes and lymphocytes due to Th1 immunity failure or loss of IFN-gamma activity in AML patients. CONCLUSIONS: We reported two cases of co-existing TB lymphadenitis and AML.TB can be the cause of febrile neutropenia in AML patients. Persistent of fever after induction chemotherapy is a clue for other additional diagnosis such as TB. Present of neutrophil in site of infection, despite of neutropenic condition, suggests inadequate function of immune response in these patients. Reference #1: Mishra P, Kumar R, Mahapatra M, et al. Tuberculosis in acute leukemia: a clinico-hematological profile. Hematology 2006;11:335-340. Reference #2: Lowe DM, Redford PS, Wilkinson RJ et al. Neutrophils in tuberculosis: friend or foe? Trends Immunol 2012;33:14-25 DISCLOSURES: No relevant relationships by Viboon Boonsarngsuk, source=Web Response No relevant relationships by Tananchai Petnak, source=Web Response

Memory-Like Natural Killer Cells

Natural killer (NK) cells, classically considered to be part of the innate immune system, are specialized for rapid responses that protect the host against pathogens and eliminate malignant cells. These functional responses are multi-faceted, and include not only direct target cell killing, but also production of cytokines and chemokines, proliferative expansion, and cross-talk with various immune cells to help orchestrate cellular immunity. NK cell recognition of a target cell is controlled by the integration of signals from germline-DNA encoded activating, inhibitory, and cytokine receptors. NK cell tolerance toward healthy host cells arises via education or licensing that requires self-inhibitory receptor engagement. Over the past decade, it has become clear that NK cells have the capacity to remember prior activation events, including hapten-exposure,1 viral infection,2 and combined cytokine stimulation.3 These studies have defined “memory”, “adaptive”, and “memory-like” responses by NK cells within both murine and human immune systems, which can result in long-lived NK cell populations with enhanced effector functionality.4-6 While certain types of NK cell “memory” can be specific to the original stimulation, other types of “memory-like” responses are non-specific, providing flexibility in the enhanced response to subsequent activating triggers.7 Cytokine-induced memory-like NK cells were originally discovered in mice, following a brief exposure to the potent activating combination of IL-12, IL-15 and IL-18.3 After this activation, murine memory-like NK cells differentiated in vivo, and demonstrated enhanced IFN-γ recall responses to IL-12 and IL-15 re-stimulation, even after extensive cell division. Subsequent studies identified human IL-12/15/18-induced memory-like NK cells that displayed enhanced function after re-stimulation via cytokine receptors, activating receptors, or tumor targets.8 Adoptive transfer of murine memory-like NK cells into syngeneic or immunodeficient mice resulted in an enhanced ability to control lymphomas and solid tumors in vivo,9 as well as the ability to persist in the recipient for months. In addition, memory-like NK cell differentiation restored the anti-tumor function of unlicensed NK cells, demonstrating that cytokine receptor signals can overcome a lack of NK cell education.10 Other studies showed that memory-like NK cells can ignore inhibitory receptor signals, and have enhanced anti-leukemia responses.11 Based on these pre-clinical findings, we translated allogeneic memory-like NK cells into the clinic in a first-in-human adoptive cell therapy trial for patients with relapsed/refractory (rel/ref) myeloid malignancies. This study demonstrated that rel/ref AML patients were able to safely receive IL-12/15/18-activated donor NK cells (up to 10x106/kg) without developing cytokine release syndrome, neurotoxicity, or graft-versus-host disease. Immune monitoring revealed that memory-like NK cells expanded, trafficked to the bone marrow, and exhibited enhanced anti-leukemia function ex vivo. Clinical responses (CR/CRi) were observed in >50% of patients with active rel/ref AML.11 Ongoing studies are exploring memory-like NK cell adoptive immunotherapy in a phase 2 trial for rel/ref AML, combined with same-donor allogeneic hematopoietic cell transplantation (HCT), and for relapse after allogeneic HCT. Active areas of investigation in memory-like NK cell biology and therapeutics include defining mechanisms that regulate memory-like differentiation and enhanced function, elucidating memory-like NK cell checkpoints, evaluating autologous memory-like NK cell responses against cancers, and developing strategies to enhance memory-like NK cell targeting of resistant malignancies.O'Leary JG, Goodarzi M, Drayton DL, Yu H, von Andrian UH. T cell- and B cell-independent adaptive immunity mediated by natural killer cells. Nat Immunol. 2006;7(5):507-16.Sun JC, Beilke JN, Lanier LL. Adaptive immune features of natural killer cells. Nature. 2009;457(7229):557-61.Cooper MA, Elliott JM, Keyel PA, et al. Cytokine-induced memory-like natural killer cells. Proc Natl Acad Sci USA. 2009;106(6):1915-9.Rölle A, Pollmann J, Cerwenka A. Memory of Infections: An Emerging Role for Natural Killer Cells. PLoS Pathog. 2013;9(9):1-3.Schlums H, Cichocki F, Tesi B, et al. Cytomegalovirus Infection Drives Adaptive Epigenetic Diversification of NK Cells with Altered Signaling and Effector Function. Immunity. 2015;42(3):443-456.Lee J, Zhang T, Hwang I, et al. Epigenetic Modification and Antibody-Dependent Expansion of Memory-like NK Cells in Human Cytomegalovirus-Infected Individuals. Immunity. 2015;42(3):431-442.Fehniger TA, Cooper MA. Harnessing NK Cell Memory for Cancer Immunotherapy. Trends Immunol. 2016;Epub Oct 2:10.1016/j.it.2016.09.005.Romee R, Schneider SE, Leong JW, et al. Cytokine activation induces human memory-like NK cells. Blood. 2012;120(24):4751-4760.Ni J, Miller M, Stojanovic A, Garbi N, Cerwenka A. Sustained effector function of IL-12/15/18-preactivated NK cells against established tumors. J Exp Med. 2012;209(13):2351-2365.Wagner JA, Berrien-Elliott MM, Rosario M, et al. Cytokine-Induced Memory-Like Differentiation Enhances Unlicensed NK Cell Anti-Leukemia and FcγRIIIa-Triggered Responses. Biol. Blood Marrow Transplant. 2016;dx.doi.org:Romee R, Rosario M, Berrien-Elliott MM, et al. Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukemia. Sci. Transl. Med. 2016;8(357):357:doi: 10.1126/scitranslmed.aaf2341. DisclosuresFehniger:Altor BioScience: Research Funding; Cyto-Sen Therapeutics: Consultancy; Celgene: Research Funding; NIH/NCI: Other: R01 CA205239, P50CA171963; Affimed: Research Funding.

New Flavors in Immunomodulation

New Flavors in Immunomodulation

Abstract PL1: Potential of Radiation Therapy to Convert the Tumor Into an In Situ Vaccine

Abstract Radiotherapy (RT) has revealed an ideal adjuvant to cancer immunotherapy, because of it's ability to convert the irradiated tumor into an individualized, in situ vaccine. 1 DNA damage response (DDR) contributes to immune rejection of tumors, a mechanism at least in part responsible of the success of platinum and RT combinations. 2 RT-induced cell death can evoke T cell memory, inducing effects outside the irradiated field, defined as abscopal. In the setting of metastatic cancer, however, the occurrence of abscopal effects is extremely rare, 3 because of the established immune-suppressive microenvironment. 4 Thus, the pro-immunogenic effects of radiotherapy is best exploited in combination with immunotherapy, 5,6 and combination of RT and immune checkpoint blockade (ICB) has matured to reach clinical translation. 7 8 The issue of RT dose and fractionation is particularly relevant to abscopal responses. 8,9 A mechanism underlying this dose-dependence was recently elucidated. 10 In mice bearing bilateral TSA murine breast carcinoma a single dose of 20 or 30Gy combined with ICB achieved comparable in field control to that of a regimen of 8GyX3 fractions, but only the fractionated regimen induced abscopal responses. An interferon type I (IFN-I) gene signature was associated with the 8GyX3 fractions but not with a single dose of 20 or 30 Gy. IFN-I plays a key role in cross priming and rejection of cancer. 11 RT-generates double strands (ds) DNA fragments reach the cytoplasm of irradiated cells where they are “sensed” by cGAS/STING (cGAS=cyclic GMP-AMP synthase and its adaptor protein STING= stimulator of interferon genes, aka transmembrane protein 173 – TMEM173). cGAS binds cytosolic dsDNA to initiate IFN-I responses upon STING stimulation, resulting in dendritic cell recruitment and cross-priming of effector T-cells, key steps to convert the tumor into an in situ vaccine. In multiple carcinoma murine and human carcinoma cells as the radiation dose increased, cytosolic dsDNA was found to accumulate to a threshold above which induction of three prime repair exonuclease 1 (Trex1) occured, an enzyme that degrades cytoplasmic DNA. Single doses in excess of 10-12 Gy induced Trex1, with rapid degradation of cytosolic dsDNA, and abrogation of IFN induction, impairing RT-induced abscopal effects. These findings support the choice of 3-5 doses of 8 Gy each, when radiotherapy is combined with immunotherapy. Interestingly, a recent dose escalation clinical trial demonstrated an inferior incidence of pathological response (pCR) when breast cancers were irradiated beyond a certain threshold: pCR was 67% in the cohort treated with 8.5 Gy X3 and decreased to 43 and 33% after three fractions of 9.5Gy and 10.5 Gy, respectively. 12 Ongoing investigations are defining optimal sequencing and treatment fields. 1. OncoImmunology 2014; 3: e28518. 2. Oncoimmunology 2017; 6(3): e1274479. 3. Curr Probl Cancer 2016; 40: 25-37. 4. Annu Rev Immunol 2011; 29: 235-71. 5. Int J Radiat Oncol Biol Phys 2004; 58: 862-70. 6. Lancet Oncol 2015; 16(7): 795-803. 7. Clin Cancer Res 2005; 11: 728-34. 8. J Immunother Cancer 2016; 4: 51. 9. Clin Cancer Res 2009; 15(17): 5379-88. 10. Nat Commun 2017; 8: 15618. 11.Trends Immunol 2013; 34(2): 67-73. 12. Int J Radiat Oncol Biol Phys 2013; 85(5): 1193-9. Citation Format: Formenti SC. Potential of Radiation Therapy to Convert the Tumor Into an In Situ Vaccine [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PL1.

Research Highlights

Research Highlights

IL-17-Driven Immune Signaling in Autoimmunity and Fungal Infections

Interleukin (IL)-17 is a pro-inflammatory cytokine that is the founding member of a distinct subclass of cytokines (reviewed in Amatya et al., 2017). IL0-17 is produced by conventional Th17 cells as well as a variety of innate immune subtypes (gd-T cells, ILC3 and NKT cells, for example). IL-17 promotes immunity to extracellular microbes, particularly fungi. On the flip side, dysregulation of IL-17 expression or signaling promotes pathology in a variety of autoimmune settings, particularly plaque psoriasis. Consequently, biologic therapies targeting the IL-17/Th17 pathway have been used successfully in the clinic. Understanding how the balance between host defense and autoimmune inflammation is maintained remains a vital issue. Candida albicans is a commensal fungus responsible for opportunistic oropharyngeal candidiasis (OPC, thrush) in settings of immunodeficiency such as HIV/AIDS and chemotherapy. C. albicans also causes systemic and dermal infections. In recent years it has become evident that IL-17 is essential for protective immunity to C. albicans (Conti and Gaffen, 2015). In both mice and humans, T cell deficiencies and more specifically impairments in IL-17 receptor (IL-17R) signaling components (for example, IL-17RA, IL-17RC, Act1) are associated with chronic mucosal candidiasis. However, to date we lack a detailed understanding of the signaling components and effector activities of IL-17 that promote antifungal immunity. The IL-17R complex is ubiquitously expressed, and antifungal signaling by IL-17 has been documented in cells of mesenchymal and epithelial origin but also in hematopoietic cells. This talk will focus on multiple mechanisms by which IL-17 drives immunity to C. albicans, including (i) tissue-specific effects of IL-17 signaling, (ii) sources of IL-17 that drive immunity to mucosal candidiasis, and (iii) fungal-derived factors that drive IL-17 production in vivo in the context of infection.Amatya N, Garg AV, Gaffen SL. IL-17 signaling: The Yin and the Yang. Trends Immunol. 2017; 38:310-322.Conti HR, Gaffen SL. IL-17 and Candida albicans infections. J Immunol, 2015; 195:780-788. DisclosuresGaffen:Lycera: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Abbvie: Consultancy; UCB Celltech: Consultancy; Genentech: Speakers Bureau; Novartis: Consultancy.

Research Highlights

Research Highlights

Attacking the system: Next-generation auto-immune therapies target pathways rather than symptoms.

Auto‐immune diseases are a major health problem in both developed and developing countries: They cause as many deaths as the leading infectious diseases and exact an even greater toll on patients’ quality of life given their chronic nature (Box 1). Reliable data for all auto‐immune diseases are hard to collect, but a 2011 study by the American Autoimmune Related Diseases Association (AARDA) reported that the total annual cost of just seven leading auto‐immune diseases (Crohn's disease, ulcerative colitis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), rheumatoid arthritis (RA), psoriasis, and scleroderma) was somewhere between US$51.8 billion and US$70.6 billion in the USA alone (http://www.diabetesed.net/page/_ files/autoimmune-diseases.pdf). The AARDA report also drew attention to the indirect societal toll with one example being sufferers from RA in the USA, who experienced a decline in average earnings from US$18,409 to US$13,900 per year, and the number of jobs they were able to perform dropped from 11.5 to 2.6 million. It was also found that approximately 50 percent of RA patients were unable to work at all within ten years after disease onset. ### Box 1: Some common auto‐immune diseases #### Graves’ Disease The most common auto‐immune disease in which the body produces antibodies to the receptor for thyroid‐stimulating hormone (TSH). It usually attacks the thyroid, frequently causing it to enlarge to twice its size or more (goiter) and become overactive, with related hyperthyroid symptoms such as increased heartbeat, muscle weakness, disturbed sleep, and irritability. No ideal cure but it can be controlled, with last resort surgical excision of the gland. Other treatments include anti‐thyroid drugs, which reduce the production of thyroid hormone, and radioiodine to shrink the gland and reduce its activity. #### Hashimoto's thyroiditis Another auto‐immune disease of the thyroid gland that involves a variety of cell‐ and antibody‐mediated immune processes. It was the first disease to be recognized as auto‐immune in 1912. In this case, the …

Histone Deacetylase 11 (HDAC11) As a Novel Transcriptional Regulator of C/EBP-β, in Immature Myeloid Cell to Myeloid Derived Suppressor Cell Transition

In normal myelopoiesis, immature myeloid cells (IMCs) differentiate into macrophages, dendritic cells, and neutrophils. However, in pathological conditions such as in cancer, these immature cells differentiate into myeloid derived suppressor cells (MDSCs). Transcription factor CCAAT-enhancer-binding protein beta (C/EBP-β) plays a crucial role in the accumulation of MDSCs in several pathological conditions. Of note, mice lacking C/EBP-β in the bone marrow compartment lose the ability to differentiate IMCs into pathologically active MDSCs2.In contrast, up-regulation of C/EBP-β can partly induce MDSC expansion through a mechanism involving the STAT3 pathway3.Epigenetic changes, such as histone acetylation status, have been recently shown to modulate the regulatory function of MDSCs4. However, the epigenetic mechanism(s) involved in regulation of C/EBP-β gene expression in MDSCs are poorly understood. Here we show for the first time that among all the HDACs known, HDAC11, the newest member of this family of enzymes, is recruited to the C/EBP-β gene promoter region of primary myeloid cells isolated from C57BL/6 mice. Such a finding led us to explore the functional consequences of disrupting HDAC11 in myeloid cells. First, we observed a markedly higher expression of C/EBP-β mRNA (194 fold difference) in the CD11b+/Ly6G+ granulocytic compartment and a 6.7 fold difference in the CD11b+/Ly6C+monocytic compartments of HDAC11KO mice relative to control wild type mice. Second, MDSCs from HDAC11 KO mice display a higher suppressive phenotype and mechanistically we have found that they have increased expression of the immunosuppressive STAT3/IL10 axis. Third, inoculation of EL4 tumor cells into HDAC11KO mice and controls resulted in a higher expansion capacity of splenic MDSCs in mice devoid of HDAC11. Such an effect was associated with a more rapid tumor growth in HDAC11KO mice as compared to WT mice. Similar aggressive tumor growth was also observed in HDAC11 myeloid conditional KO mice (LyZ-Cre-HDAC11KO).Taken together, we have uncovered a previously unknown role for HDAC11 as a transcriptional regulator of C/EBP-β in MDSCs. Indeed, in the absence of this epigenetic checkpoint of C/EBP-β gene expression, the fully suppressive potential of MDSCs was fully unleashed. A better understanding of this novel role of HDAC11 in myeloid biology will ultimately lead to targeted epigenetic therapies to manipulate the suppressive abilities of these immunoregulatory cells.1. Hirai H, Zhang P, Dayaram T, et al. C/EBPbeta is required for 'emergency’ granulopoiesis. Nat Immunol 2006; 7(7): 732-9.2. Marigo I, Bosio E, Solito S, et al. Tumor-induced tolerance and immune suppression depend on the C/EBPbeta transcription factor. Immunity 2010; 32(6): 790-802.3. Condamine T, Gabrilovich DI. Molecular mechanisms regulating myeloid-derived suppressor cell differentiation and function. Trends Immunol 2011; 32(1): 19-25.4. Youn JI, Kumar V, Collazo M, et al. Epigenetic silencing of retinoblastoma gene regulates pathologic differentiation of myeloid cells in cancer. Nat Immunol 2013; 14(3): 211-20. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2542: Sipuleucel-T-induced immune response against secondary cancer antigens is associated with improved overall survival

Abstract Introduction and Objective: Tumor cell death in response to immunotherapy may lead to the release of secondary (non-targeted) cancer antigens that prime subsequent immune responses (antigen spread) 1. This may contribute to tumor control and reveal post-treatment biomarkers of clinical outcome. Sipuleucel-T, an immunotherapy targeted towards the prostate-specific antigen, prostatic acid phosphatase, is approved in the US and EU for treatment of asymptomatic or minimally symptomatic, metastatic castration-resistant prostate cancer. We evaluated: (i) antigen spread after sipuleucel-T treatment, based on IgG antibody responses against secondary cancer antigens, and (ii) association of such IgG responses with overall survival (OS). Methods: ProtoArray®, an unbiased protein microarray platform, was used to characterize the spectrum of serum IgG responses after treatment. IgG responses against secondary antigens were confirmed using an independent platform, Luminex®, xMAP®. Pre- and post- treatment sera from two clinical trials of sipuleucel-T were analyzed: IMPACT 2 (double-blind, placebo-controlled, phase 3, NCT00065442) and ProACT (open-label, phase 2, NCT00715078). The correlations between IgG responses (≥ 2 fold elevation post-treatment) and OS were assessed in IMPACT using a Cox regression model, adjusted for baseline PSA, LDH, bone lesions, Gleason score, and prior bisphosphonate use. Results: Serum IgG responses (p&amp;lt;10-3) towards several secondary cancer antigens (PSA, hK2/KLK2, K-Ras, E-Ras, and LGALS8/PCTA-1) were observed after treatment with sipuleucel-T in IMPACT as well as ProACT. Across trials, the frequency of patients with IgG responses after sipuleucel-T treatment was: ≥25% for PSA, ≥33% for hK2, ≥36% for K-Ras, ≥39% for E-Ras, ≥24% for LGALS8. These IgG responses were observed as early as 2 weeks and up to 6 months after treatment with sipuleucel-T. Control arm patients in IMPACT did not show such responses (p&amp;gt;0.01). In the sipuleucel-T arm of IMPACT (N=140), an IgG response to PSA ≈2 weeks after treatment was positively correlated with increased OS, compared to patients with no IgG response to PSA (Cox p&amp;lt;0.01, HR≈0.41 [95% CI=0.22-0.78]). Other IgG responses (such as those against E-Ras and hK2) at ≈2 or ≈10 weeks after treatment, also showed trends towards improved OS. Conclusions: Sipuleucel-T elicited IgG responses against multiple cancer antigens. Sipuleucel-T- induced IgG response against PSA was positively correlated with improved OS. These data enhance our understanding of sipuleucel-T's mechanism of action, and may help to identify pharmacodynamic biomarkers of clinical outcome. The methods and results presented here may also help in the characterization of antigen spread and pharmacodynamic biomarkers after treatment with other cancer immunotherapies. 1. Ribas A, et al. Trends Immunol. 24:58-61 (2003) 2. Kantoff PW, et al. N Engl J Med. 363:411-22 (2010) Citation Format: Debraj GuhaThakurta, Li-Qun Fan, Tuyen Vu, Francis Stewart, Philip Kantoff, Eric Small, Celeste Higano, Thomas Gardner, Nadeem Sheikh, James Trager. Sipuleucel-T-induced immune response against secondary cancer antigens is associated with improved overall survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2542. doi:10.1158/1538-7445.AM2014-2542

AB0175 C1Q Regulates the TLR7-Mediated Inflammatory Response by Pristane-Primed Macrophages: Implications for Pristane-Induced Lupus (PIL)

BackgroundIntraperitoneal injection of the natural oil pristane induces loss of immune tolerance and development of pristane-induced lupus (PIL) in susceptible mouse strains. PIL is characterised by high titres of autoantibodies,...