Characterization of ultrapotent chemogenetic ligands for research applications in non-human primates

Chemogenetics is a technique for obtaining selective pharmacological control over a cell population by expressing an engineered receptor that is selectively activated by an exogenously administered ligand. A promising approach for neuronal modulation involves the use of Pharmacologically Selective Actuator Modules (PSAMs); these chemogenetic receptors are selectively activated by ultrapotent Pharmacologically Selective Effector Molecules (uPSEMs). To extend the use of PSAM/PSEMs to studies in nonhuman primates it is necessary to thoroughly characterize the efficacy and safety of these tools. We describe the time course and brain penetrance in rhesus monkeys of two compounds with promising binding specificity and efficacy profiles in in vitro studies, uPSEM792 and uPSEM817, after systemic administration. Rhesus macaques received subcutaneous (s.c.) or intravenous (i.v.) administration of uPSEM817(0.064 mg/kg) or uPSEM792 (0.87 mg/kg) and plasma and CSF samples were collected over the course of 48 hours. Both compounds exhibited good brain penetrance, relatively slow washout and negligible conversion to potential metabolites - varenicline or hydroxyvarenicline. In addition, we found that neither of these uPSEMs significantly altered heart rate or sleep. Our results indicate that both compounds are suitable candidates for neuroscience studies using PSAMs in nonhuman primates.

Predication of the Effector Proteins Secreted by Fusarium sacchari Using Genomic Analysis and Heterogenous Expression

One of the causative agents of pokkah boeng disease (PBD), which affects sugarcane crops globally, is the fungus Fusarium sacchari. These fungal infections reduce sugar quality and yield, resulting in severe economic losses. Effector proteins play important roles in the interactions between pathogenic fungi and plants. Here, we used bioinformatic prediction approaches to identify 316 candidate secreted effector proteins (CSEPs) in the complete genome of F. sacchari. In total, 95 CSEPs contained known conserved structures, representing 40 superfamilies and 18 domains, while an additional 91 CSEPs contained seven known motifs. Of the 130 CSEPs containing no known domains or motifs, 14 contained one of four novel motifs. A heterogeneous expression system in Nicotiana benthamiana was used to investigate the functions of 163 CSEPs. Seven CSEPs suppressed BAX-triggered programmed cell death in N. benthamiana, while four caused cell death in N. benthamiana. The expression profiles of these eleven CSEPs during F. sacchari infection suggested that they may be involved in sugarcane-F. sacchari interaction. Our results establish a basis for further studies of the role of effector molecules in pathogen–sugarcane interactions, and provide a framework for future predictions of pathogen effector molecules.

Immune Cell-Derived Extracellular Vesicles – New Strategies in Cancer Immunotherapy

Immune cell-derived extracellular vesicles (EVs) have increasingly become the focus of research due to their unique characteristics and bioinspired applications. They are lipid bilayer membrane nanosized vesicles harboring a range of immune cell-derived surface receptors and effector molecules from parental cells. Immune cell-derived EVs are important mediators of intercellular communication that regulate specific mechanisms of adaptive and innate immune responses. However, the mechanisms underlying the antitumor effects of EVs are still being explored. Importantly, immune cell-derived EVs have some unique features, including accessibility, storage, ability to pass through blood-brain and blood-tumor barriers, and loading of various effector molecules. Immune cell-derived EVs have been directly applied or engineered as potent antitumor vaccines or for the diagnosis of clinical diseases. More research applications involving genetic engineering, membrane engineering, and cargo delivery strategies have improved the treatment efficacy of EVs. Immune cell-derived EV-based therapies are expected to become a separate technique or to complement immunotherapy, radiotherapy, chemotherapy and other therapeutic modalities. This review aims to provide a comprehensive overview of the characteristics and functions of immune cell-derived EVs derived from adaptive (CD4+ T, CD8+ T and B cells) and innate immune cells (macrophages, NK cells, DCs, and neutrophils) and discuss emerging therapeutic opportunities and prospects in cancer treatment.

Mitochondrial-Linked De Novo Pyrimidine Biosynthesis Dictates Human T-Cell Proliferation but Not Expression of Effector Molecules

T-cell activation upon antigen stimulation is essential for the continuation of the adaptive immune response. Impairment of mitochondrial oxidative phosphorylation is a well-known disruptor of T-cell activation. Dihydroorotate dehydrogenase (DHODH) is a component of the de novo synthesis of pyrimidines, the activity of which depends on functional oxidative phosphorylation. Under circumstances of an inhibited oxidative phosphorylation, DHODH becomes rate-limiting. Inhibition of DHODH is known to block clonal expansion and expression of effector molecules of activated T cells. However, this effect has been suggested to be caused by downstream impairment of oxidative phosphorylation rather than a lower rate of pyrimidine synthesis. In this study, we successfully inhibit the DHODH of T cells with no residual effect on oxidative phosphorylation and demonstrate a dose-dependent inhibition of proliferation of activated CD3+ T cells. This block is fully rescued when uridine is supplemented. Inhibition of DHODH does not alter expression of effector molecules but results in decreased intracellular levels of deoxypyrimidines without decreasing cell viability. Our results clearly demonstrate the DHODH and mitochondrial linked pyrimidine synthesis as an independent and important cytostatic regulator of activated T cells.

Involvement of Innate Immune Receptors in the Resolution of Acute Hepatitis B in Woodchucks

The antiviral property of small agonist compounds activating pattern recognition receptors (PRRs), including toll-like and RIG-I receptors, have been preclinically evaluated and are currently tested in clinical trials against chronic hepatitis B (CHB). The involvement of other PRRs in modulating hepatitis B virus infection is less known. Thus, woodchucks with resolving acute hepatitis B (AHB) after infection with woodchuck hepatitis virus (WHV) were characterized as animals with normal or delayed resolution based on their kinetics of viremia and antigenemia, and the presence and expression of various PRRs were determined in both outcomes. While PRR expression was unchanged immediately after infection, most receptors were strongly upregulated during resolution in liver but not in blood. Besides well-known PRRs, including TLR7/8/9 and RIG-I, other less-characterized receptors, such as IFI16, ZBP1/DAI, AIM2, and NLRP3, displayed comparable or even higher expression. Compared to normal resolution, a 3–4-week lag in peak receptor expression and WHV-specific B- and T-cell responses were noted during delayed resolution. This suggested that PRR upregulation in woodchuck liver occurs when the mounting WHV replication reaches a certain level, and that multiple receptors are involved in the subsequent induction of antiviral immune responses. Liver enzyme elevations occurred early during normal resolution, indicating a faster induction of cytolytic mechanisms than in delayed resolution, and correlated with an increased expression of NK-cell and CD8 markers and cytolytic effector molecules. The peak liver enzyme level, however, was lower during delayed resolution, but hepatic inflammation was more pronounced and associated with a higher expression of cytolytic markers. Further comparison of PRR expression revealed that most receptors were significantly reduced in woodchucks with established and progressing CHB, and several RNA sensors more so than DNA sensors. This correlated with a lower expression of receptor adaptor and effector molecules, suggesting that persistent, high-level WHV replication interferes with PRR activation and is associated with a diminished antiviral immunity based on the reduced expression of immune cell markers, and absent WHV-specific B- and T-cell responses. Overall, the differential expression of PRRs during resolution and persistence of WHV infection emphasizes their importance in the ultimate viral control during AHB that is impaired during CHB.

Viability Status-Dependent Effect of Bifidobacterium longum ssp. longum CCM 7952 on Prevention of Allergic Inflammation in Mouse Model

The classical definition of probiotics states that bacteria must be alive to be beneficial for human organism. However, recent reports show that inactivated bacteria or their effector molecules can also possess such properties. In this study, we investigated the physical and immunomodulatory properties of four Bifidobacterium strains in the heat-treated (HT) and untreated (UN) forms. We showed that temperature treatment of bacteria changes their size and charge, which affects their interaction with epithelial and immune cells. Based on the in vitro assays, we observed that all tested strains reduced the level of OVA-induced IL-4, IL-5, and IL-13 in the spleen culture of OVA-sensitized mice. We selected Bifidobacterium longum ssp. longum CCM 7952 (Bl 7952) for further analysis. In vivo experiments confirmed that untreated Bl 7952 exhibited allergy-reducing properties when administered intranasally to OVA-sensitized mice, which manifested in significant suppression of airway inflammation. Untreated Bl 7952 decreased local and systemic levels of Th2 related cytokines, OVA-specific IgE antibodies and simultaneously inhibited airway eosinophilia. In contrast, heat-treated Bl 7952 was only able to reduce IL-4 levels in the lungs and eosinophils in bronchoalveolar lavage, but increased neutrophil and macrophage numbers. We demonstrated that the viability status of Bl 7952 is a prerequisite for the beneficial effects of bacteria, and that heat treatment reduces but does not completely abolish these properties. Further research on bacterial effector molecules to elucidate the beneficial effects of probiotics in the prevention of allergic diseases is warranted.

OP0072 SINGLE CELL SEQUENCING REVEALS CLONALLY EXPANDED CYTOTOXIC CD4+ T CELLS IN THE JOINTS OF ACPA+ RA PATIENTS

Background:CD4+ T cells with cytotoxic functions (CD4+ CTL) have gained attention in recent years. Accumulating evidence supports their importance in defense against human viral infections such as CMV1, EBV2, dengue3, HIV4, 5 and SARS-CoV-26. Moreover, expansion of so called CD28null cytotoxic CD4+ T cells have been reported in the blood of patients with rheumatic diseases such as rheumatoid arthritis (RA)7, myositis8 and vasculitis9 as well as in cardiovascular diseases10.Objectives:Here, we aimed to investigate the presence and clonal expansion of CD4+ CTL in the peripheral blood (PB) and synovial fluid (SF) of RA patients using single cell technologies.Methods:We assessed the expression of cytotoxic effector molecules and transcription factors in CD4+ T cells in synovial fluid (n=21) and paired peripheral blood (n=16) from ACPA- and APCA+ RA patients by multi-parameter flow cytometry. We performed single cell sequencing, in combination with 5´ TCRab sequencing, on purified CD4+ T cells from the peripheral blood (PB) and synovial fluid (SF) of ACPA+ RA patients (n=7).Results:Flow cytometry experiments show that Granzyme-B+ Perforin-1+ CD4+ CTL are significantly increased in the SF of ACPA+ RA patients as compared to ACPA- RA patients (p=0.0072). The presence of CD4+ CTL could be confirmed by single cell sequencing in SF of each ACPA+ RA patient tested (n=7). Moreover, we found that the adhesion G-protein coupled receptor GPR56 is selectively expressed on the recently described peripheral helper (TPH) T-cell subset11 and associates with the expression of tissue resident memory markers LAG-3, CXCR6 and CD69. In blood, we confirmed a previous report12 showing that GPR56 delineates cytotoxic CD4+ T cells. Finally, expanded TCR clones expressing cytotoxic effector molecules were identified in synovial fluid of ACPA+ RA patients and, for some patients, in their corresponding peripheral blood.Conclusion:We identified GPR56 as a marker of TPH cells in SF of ACPA+ RA patients that associates with tissue residency receptors. The combination of single cell sequencing and multi-parameter flow cytometry highlights the importance of CD4+ CTL in ACPA+ RA and suggests a potential therapeutic target (Figure 1).References:[1]Casazza J. P. et al., J Exp Med2006,203 (13), 2865-77.[2]Landais E. et al., Blood2004,103 (4), 1408-16.[3]Kurane I. et al. J Exp Med1989,170 (3), 763-75.[4]Appay V. et al. J Immunol2002,168 (11), 5954-8.[5]Juno J. A. et al. Front Immunol2017,8, 19.[6]Meckiff B. J. et al. Cell2020,183 (5), 1340-1353 e16.[7]Schmidt D. et al. J Clin Invest1996,97 (9), 2027-37.[8]Fasth A. E. et al. J Immunol2009,183 (7), 4792-9.[9]Moosig F. et al. Clin Exp Immunol1998,114 (1), 113-8.[10]Sato K. et al. J Exp Med2006,203 (1), 239-50.[11]Rao D. A., et al. Nature2017,542 (7639), 110-114.[12]Peng Y. M. et al. J Leukoc Biol2011,90 (4), 735-40.Acknowledgements:We thank the patients who donated samples and the medical staff at the Rheumatology Clinic of Karolinska University Hospital. Julia Boström, Gloria Rostvall, and Susana Hernandez Machado are acknowledged for organizing the sampling, storage, and administration of biomaterial. This study is supported by grants from Dr. Margaretha Nilssons, the Nanna Svartz, the Ulla and Gustaf af Ugglas foundations and the Swedish association against rheumatism.Disclosure of Interests:Alexandra Argyriou: None declared, Marc H Wadsworth II Employee of: Pfizer, Inc, Cambridge, MA 02139, United States, Adrian Lendvai: None declared, Stephen Christensen Employee of: Pfizer, Inc, Cambridge, MA 02139, United States, Aase Hensvold: None declared, Christina Gerstner: None declared, Kellie Kravarik Employee of: Pfizer, Inc, Cambridge, MA 02139, United States, Aaron Winkler Employee of: Pfizer, Inc, Cambridge, MA 02139, United States, Vivianne Malmström: None declared, Karine Chemin: None declared