CD40 Ligand Research Articles

Blocking the CD47-SIRPα interaction reverses the disease phenotype in a polycythemia vera mouse model

Polycythemia vera (PV) is a hematopoietic stem cell neoplasm driven by somatic mutations in JAK2, leading to increased red blood cell (RBC) production uncoupled from mechanisms that regulate physiological erythropoiesis. At steady-state, bone marrow macrophages promote erythroid maturation, whereas splenic macrophages phagocytose aged or damaged RBCs. The binding of the anti-phagocytic (“don’t eat me”) CD47 ligand expressed on RBCs to the SIRPα receptor on macrophages inhibits phagocytic activity protecting RBCs from phagocytosis. In this study, we explore the role of the CD47-SIRPα interaction on the PV RBC life cycle. Our results show that blocking CD47-SIRPα in a PV mouse model due to either anti-CD47 treatment or loss of the inhibitory SIRPα-signal corrects the polycythemia phenotype. Anti-CD47 treatment marginally impacted PV RBC production while not influencing erythroid maturation. However, upon anti-CD47 treatment, high-parametric single-cell cytometry identified an increase of MerTK+ splenic monocyte-derived effector cells, which differentiate from Ly6Chi monocytes during inflammatory conditions, acquire an inflammatory phagocytic state. Furthermore, in vitro, functional assays showed that splenic JAK2 mutant macrophages were more “pro-phagocytic,” suggesting that PV RBCs exploit the CD47-SIRPα interaction to escape innate immune attacks by clonal JAK2 mutant macrophages.

Transcriptomic analysis of B cells suggests that CD70 and LY9 may be novel features in patients with systemic lupus erythematosus

Dysfunction of B-cell subsets is critical in the development of systemic lupus erythematosus (SLE). There is a great diversity of B-lineage cells, and their features and functions in SLE need to be clarified. In this study, we analyzed single-cell RNA sequencing (scRNA-seq) data from peripheral blood mononuclear cells (PBMCs) and bulk transcriptomic data of isolated B-cell subsets from patients with SLE and healthy controls (HCs). We preformed scRNA-seq analysis focused on the diversity of B-cell subsets and identified a subset of antigen-presenting B cells in SLE patients that highly expressed ITGAX. A list of marker genes of each B-cell subset in patients with SLE was also identified. Comparison of bulk transcriptomic data of isolated B-cell subpopulations between SLE patients and HCs revealed the upregulated differentially expressed genes (DEGs) for each B-cell subpopulation in SLE. Common genes identified using these two methods were considered to be upregulated marker genes of B cells in SLE. The scRNA-seq data of SLE patients and HCs revealed that CD70 and LY9 were overexpressed in B cells vs. other cell types from SLE patients, and this pattern was validated by RT‒qPCR. Because CD70 is the cellular ligand of CD27, previous studies on CD70 have focused mainly on T cells from SLE patients. LY9 appears to have different functions in mice and humans: its expression is decreased in lupus-prone mice but is increased in T cells and some B-cell subpopulations in SLE patients. Here, we describe the overexpression of two costimulatory molecules, CD70 and LY9, which may be a novel feature of B cells in SLE patients.

CTLA4 depletes T cell endogenous and trogocytosed B7 ligands via cis-endocytosis.

CD28 and CTLA4 are T cell coreceptors that competitively engage B7 ligands CD80 and CD86 to control adaptive immune responses. While the role of CTLA4 in restraining CD28 costimulatory signaling is well-established, the mechanism has remained unclear. Here, we report that human T cells acquire antigen-presenting-cell (APC)-derived B7 ligands and major histocompatibility complex (MHC) via trogocytosis through CD28:B7 binding. Acquired MHC and B7 enabled T cells to autostimulate, and this process was limited cell-intrinsically by CTLA4, which depletes B7 ligands trogocytosed or endogenously expressed by T cells through cis-endocytosis. Extending this model to the previously proposed extrinsic function of CTLA4 in human regulatory T cells (Treg), we show that blockade of either CD28 or CTLA4 attenuates Treg-mediated depletion of APC B7, indicating that trogocytosis and CTLA4-mediated cis-endocytosis work together to deplete B7 from APCs. Our study establishes CTLA4 as a cell-intrinsic molecular sink that limits B7 availability on the surface of T cells, with implications for CTLA4-targeted therapy.

Abstract 3477: Maximizing the outcome of CD226 stimulation through targeting beyond TIGIT signaling with combination and multi-specific approaches for cancer immunotherapy

Abstract The CD226 (DNAM-1) axis has emerged as an important immune-modulatory pathway that regulates T cell and NK cell effector functions in the solid tumor microenvironment. Three inhibitory receptors, TIGIT, CD96, and PVRIG, inhibit the activating receptor CD226 by competing for shared ligands (CD112 and CD155) expressed on tumor cells and myeloid cells. Blocking the TIGIT receptor with monoclonal antibodies is a major focus of current clinical development efforts. However, the effectiveness of targeting TIGIT alone in a clinical setting has yet to be demonstrated. We hypothesize that blocking TIGIT alone may not lead to an optimal clinical outcome since immuno-stimulatory interaction between the ligands CD112 and CD155 with CD226 can be competed by inhibitory receptors CD96 and PVRIG in addition to TIGIT. It is therefore interesting to investigate whether all these checkpoints play critical roles in the tumor environment and if inhibiting one or more may be necessary to achieve the maximum CD226 activation in both innate and adaptive immune cells. To address that, we have conducted single-cell analysis and demonstrated high expression levels and co-expression of the three inhibitory receptors on T cell subsets and NK cells in the tumor microenvironment for multiple tumor types. Thus, simultaneous blockade of two or more of these receptors might be required for full CD226 activation and T and NK cell-mediated anti-tumor immunity. We envision developing blocking antibodies to these receptors in the CD226 axis toward potential combinations or multi-specific antibody therapies against solid tumors. Antibodies against all three checkpoints, i.e., TIGIT, CD96, and PVRIG, were selected based on receptor binding and their ability to block the receptor interaction with their ligands. We have demonstrated the functionality of the antibodies against these three targets and explored the best approach to alleviate the suppressive regulation of CD226 signaling. Detailed characterizations of these antibodies have been performed, and multi-specific antibodies have been generated and evaluated. Through this effort, we aim to define and develop the best therapeutic strategy for unleashing the CD226 axis for T and NK cell-mediated anti-tumor immunity. Citation Format: Tom Li Stephen, Qian Zhang, Byung-Kwon Lee, Chao Kong, Rain Sun, Jia Wu, Jie Cui, Hillary Shah, Wenhua Xu, Yun-Yueh Lu, Joshua Whitener, Emily Jackson, Jinping Gan, Francisco Adrian, Liang Schweizer. Maximizing the outcome of CD226 stimulation through targeting beyond TIGIT signaling with combination and multi-specific approaches for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3477.

CD30 stimulation induces multinucleation and chromosomal instability in HTLV-1-infected cell lines.

A recent report indicated involvement of CD30 in progression of human leukemia virus type 1 (HTLV-1) infection, but the exact roles of CD30 in this process remain unclear. This study was conducted to determine the role of CD30 by stimulating CD30 expressed on HTLV-1-infected cell lines with CD30 ligand and observing its effects. CD30 stimulation increased multinucleated cells and inhibited proliferation of HTLV-1-infected cells. This inhibition was recovered by interruption of CD30 stimulation. Chromatin bridges found in multinucleated cells suggested DNA damage. CD30 stimulation triggered DNA double-strand breaks (DSBs) and chromosomal imbalances. CD30 stimulation induced reactive oxygen species (ROS), which induced DSBs. Generation of ROS and multinucleated cells by CD30 was dependent on phosphoinositide 3-kinase. RNA sequencing showed that CD30 stimulation produced significant changes in gene expression profiles, including upregulation of programmed death ligand 1 (PD-L1). Tax, which has also been shown to induce multinucleation and chromosomal instability, failed to induce CD30. These results suggest that induction of CD30, independent of Tax, triggers morphological abnormalities, chromosomal instability, and alteration of gene expression in HTLV-1-infected cells.

Abstract 1346: Phagocytosis of solid tumors favors macrophage clusters and cooperativity in tumor elimination

Abstract Macrophages are abundant in many solid tumors and are potential effector cells for monoclonal antibody-based immunotherapies because they can in principle phagocytose IgG-opsonized cancer cells. However, it remains unknown whether phagocytic macrophages can overcome the cohesive forces between cancer cells in solid tumors. We show that maximum phagocytosis of B16 melanoma tumors and ‘tumoroids’ requires IgG-opsonization and very deep suppression of the macrophage checkpoint ligand CD47. Under these conditions, phagocytic macrophages cluster or ‘phase separate’, and the elimination exhibits clear cooperativity with respect to macrophage number and phagocytic activity. In vitro disruption of phagocytic clusters specifically implicates Mg2+ and macrophage integrins. Cytokine treatments and other inhibitors further implicate roles for integrins and phagocytic surface receptors. Although tumor-associated macrophages correlate with negative prognoses in many cancers, our findings are among the first to reveal advantages of high macrophage numbers, macrophage clusters, and resulting cooperative effects when combined with opsonizing antibodies and CD47 disruption. Lastly, the challenging B16 tumors are durably eliminated by the approach and induce de novo IgG's that also drive anti-cancer phagocytosis. Citation Format: Dennis E. Discher. Phagocytosis of solid tumors favors macrophage clusters and cooperativity in tumor elimination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1346.

Abstract 2983: Tumor-specific CD28 costimulatory bispecific antibodies enhance T cell activation in solid tumors

Abstract T cells in the tumor micro-environment require TCR/MHC engagement and co-stimulatory receptor engagement to achieve optimal activation. Solid tumor cells lack expression of CD28 ligands, so we hypothesized that activation of CD28 signaling at the T cell/tumor cell interface could enhance anti-tumor activity. We developed a CD28 bispecific platform that allows for the rapid generation of tumor-associated antigen (TAA) x CD28 bispecific antibodies that conditionally provide CD28 costimulation only in the presence of TAA and TCR engagement. Here, we present results for a set of bispecific antibodies with broad applicability across a range of solid tumors, including CEACAM5 x CD28, Trop-2 x CD28, STEAP1 x CD28, and mesothelin x CD28. We developed optimized CD28 scFv and Fabs that cover a range of affinities and are only agonistic in the context of TCR engagement. A matrix of bispecifics pairing these CD28 binders with CEACAM5, Trop-2, STEAP1, and mesothelin antibodies of varying affinities and epitopes were generated using Xencor’s XmAb® bispecific antibody platform. Activities of these bispecifics were assessed in vitro by measuring T cell proliferation, cytokine production, and cytotoxicity in co-cultures of human cancer cell lines mixed with primary human T cells. CEACAM5 x CD28, Trop-2 x CD28, STEAP1 x CD28, and mesothelin x CD28 bispecifics enhanced T cell degranulation, cytokine secretion, and cancer cell cytotoxicity only in concert with TCR engagement. IHC was used to compare TAA expression on cancer cell lines versus that found on cancer tissues, allowing categorization of cell lines as high density (tumor surrogate) or low density (normal tissue surrogate). CD28 bispecifics were identified with selective potency on high versus low expressing cell lines, suggesting a favorable therapeutic index. CD28 bispecific antibodies co-targeting CEACAM5, Trop-2, STEAP1, and mesothelin show promising activity and warrant further development across a range of solid tumors. Citation Format: Matthew A. Dragovich, Viralkumar Davra, Matthew S. Faber, Yoon Kyung Kim, Alex Nisthal, Veronica G. Zeng, Jonathan Jacinto, Juan E. Diaz, Thuy Truong, Jing Qi, Kendra N. Avery, Rumana Rashid, Sung-Hyung Lee, Seung Y. Chu, Christine Bonzon, Ruschelle Love, Matthew J. Bernett, James A. Ernst, Rena Bahjat, Norman J. Barlow, John R. Desjarlais, Michael Hedvat, Gregory L. Moore. Tumor-specific CD28 costimulatory bispecific antibodies enhance T cell activation in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2983.

Neutrophil Extracellular Traps and Platelet Activation for Identifying Severe Episodes and Clinical Trajectories in COVID-19

The role of NETs and platelet activation in COVID-19 is scarcely known. We aimed to evaluate the role of NETs (citrullinated histone H3 [CitH3], cell-free DNA [cfDNA]) and platelet activation markers (soluble CD40 ligand [CD40L] and P-selectin) in estimating the hazard of different clinical trajectories in patients with COVID-19. We performed a prospective study of 204 patients, categorized as outpatient, hospitalized and ICU-admitted. A multistate model was designed to estimate probabilities of clinical transitions across varying states, such as emergency department (ED) visit, discharge (outpatient), ward admission, ICU admission and death. Levels of cfDNA, CitH3 and P-selectin were associated with the severity of presentation and analytical parameters. The model showed an increased risk of higher levels of CitH3 and P-selectin for ED-to-ICU transitions (Hazard Ratio [HR]: 1.35 and 1.31, respectively), as well as an elevated risk of higher levels of P-selectin for ward-to-death transitions (HR: 1.09). Elevated levels of CitH3 (HR: 0.90), cfDNA (HR: 0.84) and P-selectin (HR: 0.91) decreased the probability of ward-to-discharge transitions. A similar trend existed for elevated levels of P-selectin and ICU-to-ward transitions (HR 0.40); In conclusion, increased NET and P-selectin levels are associated with more severe episodes and can prove useful in estimating different clinical trajectories.

Gut microbiota dysbiosis associated with different types of demyelinating optic neuritis in patients.

Demyelinating optic neuritis (DON) causes rapid vision loss in young and middle-aged people. The limited efficacy of treatment and the toxic side effects of drugs significantly affect the quality of life of patients with DON. Therefore, DON pathogenesis has always been a research hotspot in terms of prevention and treatment. Studies have suggested that gut microbiota imbalances may be involved in autoimmune disease development via the modulation of multiple inflammatory cytokines and anti-inflammatory metabolites. Therefore, this study aims to explore gut microbiota differences between healthy controls (HCs) and patients with DON. A total of 54 patients with DON and 41 HCs were recruited. Fecal and blood samples were collected before and after intravenous methylprednisolone pulse (IVMP) treatment. The Shannon index, gut microbiota structure, and differential bacteria were evaluated and compared. The Shannon diversity index was decreased in patients with DON (p < 0.001) but was higher after IVMP treatment (p < 0.05). In patients with DON, Blautia, Escherichia-Shigella, and Ruminococcus showed higher abundances, whereas Bacteroides, Faecalibacterium, Roseburia, Parabacteroides, Romboutsia, and Alistipes showed lower abundances compared to that in the HCs. After IVMP treatment, the Shannon index of the myelin oligodendrocyte glycoprotein-immunoglobulin G (+) (MOG-IgG (+)) and both aquaporin-4 (AQP4)-IgG (-) and MOG-IgG (-) groups increased (p < 0.05). Bacteroides was negatively correlated with interleukin (IL)-21, IL-17E, and tumor necrosis factor-α levels (p < 0.05, r=-0.54; p < 0.05, r= -0.50; p < 0.05, r =-0.55, respectively). Escherichia was positively correlated with macrophage inflammatory protein-3α (p < 0.05, r=0.51). Alistipes was negatively correlated with soluble CD40 ligand (p < 0.05, r=-0.52). The gut microbiota differed significantly between patients with DON and HCs; however, IVMP treatment may restore gut microbiota diversity and structure in patients with DON. Moreover, gut microbiota changes may play a role in DON pathogenesis.

Deficiency of the CD155-CD96 immune checkpoint controls IL-9 production in giant cell arteritis

Loss of function of inhibitory immune checkpoints, unleashing pathogenic immune responses, is a potential risk factor for autoimmune disease. Here, we report that patients with the autoimmune vasculitis giant cell arteritis (GCA) have a defective CD155-CD96 immune checkpoint. Macrophages from patients with GCA retain the checkpoint ligand CD155 in the endoplasmic reticulum (ER) and fail to bring it to the cell surface. CD155low antigen-presenting cells induce expansion of CD4+CD96+ Tcells, which become tissue invasive, accumulate in the blood vessel wall, and release the effector cytokine interleukin-9 (IL-9). In a humanized mouse model of GCA, recombinant human IL-9 causes vessel wall destruction, whereas anti-IL-9 antibodies efficiently suppress innate and adaptive immunity in the vasculitic lesions. Thus, defective surface translocation of CD155 creates antigen-presenting cells that deviate Tcell differentiation toward Th9 lineage commitment and results in the expansion of vasculitogenic effector Tcells.

Preclinical development of a first-in-class vaccine encoding HER2, Brachyury and CD40L for antibody enhanced tumor eradication

The induction of antiviral innate immunity by systemic immunization with live virus can be employed to positively impact the response to therapeutic vaccination. We previously demonstrated that systemic immunization with a non-replicating MVA encoding CD40 ligand (CD40L) enhances innate immune cell activation and function, and triggers potent antitumor CD8+ T cell responses in different murine tumor models. Antitumor efficacy was increased when combined with tumor targeting antibodies. Here we report the development of TAEK-VAC-HerBy (TVH), a first-in-class human tumor antibody enhanced killing (TAEK) vaccine based on the non-replicating MVA-BN viral vector. It encodes the membrane bound form of human CD40L, HER2 and the transcription factor Brachyury. TVH is designed for therapeutic use in HER2- or Brachyury-expressing cancer patients in combination with tumor targeting antibodies. To preclude possible oncogenic activities in infected cells and to prevent binding of vaccine-encoded HER2 by monoclonal antibodies trastuzumab and pertuzumab, genetic modifications of HER2 were introduced in the vaccine. Brachyury was genetically modified to prevent nuclear localization of the protein thereby inhibiting its transcriptional activity. CD40L encoded in TVH enhanced human leukocyte activation and cytokine secretion in vitro. Lastly, TVH intravenous administration to non-human primates was proven immunogenic and safe in a repeat-dose toxicity study. Nonclinical data presented here highlight TVH as a first-in-class immunotherapeutic vaccine platform currently under clinical investigation.

MD Simulation Reveals Regulation of Mechanical Force and Extracellular Domain 2 on Binding of DNAM-1 to CD155

Two extracellular domains of the adhesive receptor DNAM-1 are involved in various cellular biological processes through binding to ligand CD155, usually under a mechano-microenvironment. The first extracellular domain (D1) plays a key role in recognition, but the function of the second extracellular domain (D2) and effects of force on the interaction of DNAM-1 with CD155 remain unclear. We herein studied the interaction of DNAM-1 with CD155 by performing steered molecular dynamics (MD) simulations, and observed the roles of tensile force and D2 on the affinity of DNAM-1 to CD155. The results showed that D2 improved DNAM-1 affinity to CD155; the DNAM-1/CD155 complex had a high mechanical strength and a better mechanical stability for its conformational conservation either at pulling with constant velocity or under constant tensile force (≤100 pN); the catch-slip bond transition governed CD155 dissociation from DNAM-1; and, together with the newly assigned key residues in the binding site, force-induced conformation changes should be responsible for the mechanical regulation of DNAM-1's affinity to CD155. This work provided a novel insight in understanding the mechanical regulation mechanism and D2 function in the interaction of DNAM-1 with CD155, as well as their molecular basis, relevant transmembrane signaling, and cellular immune responses under a mechano-microenvironment.

Occupational quartz and particle exposure affect systemic levels of inflammatory markers related to inflammasome activation and cardiovascular disease

BackgroundThe inflammatory responses are central components of diseases associated with particulate matter (PM) exposure, including systemic diseases such as cardiovascular diseases (CVDs). The aim of this study was to determine if exposure to PM, including respirable dust or quartz in the iron foundry environment mediates systemic inflammatory responses, focusing on the NLRP3 inflammasome and novel or established inflammatory markers of CVDs.MethodsThe exposure to PM, including respirable dust, metals and quartz were determined in 40 foundry workers at two separate occasions per worker. In addition, blood samples were collected both pre-shift and post-shift and quantified for inflammatory markers. The respirable dust and quartz exposures were correlated to levels of inflammatory markers in blood using Pearson, Kendall τ and mixed model statistics. Analyzed inflammatory markers included: 1) general markers of inflammation, including interleukins, chemokines, acute phase proteins, and white blood cell counts, 2) novel or established inflammatory markers of CVD, such as growth/differentiation factor-15 (GDF-15), CD40 ligand, soluble suppressor of tumorigenesis 2 (sST2), intercellular/vascular adhesion molecule-1 (ICAM-1, VCAM-1), and myeloperoxidase (MPO), and 3) NLRP3 inflammasome-related markers, including interleukin (IL)-1β, IL-18, IL-1 receptor antagonist (IL-1Ra), and caspase-1 activity.ResultsThe average respirator adjusted exposure level to respirable dust and quartz for the 40 foundry workers included in the study was 0.65 and 0.020 mg/m3, respectively. Respirable quartz exposure correlated with several NLRP3 inflammasome-related markers, including plasma levels of IL-1β and IL-18, and several caspase-1 activity measures in monocytes, demonstrating a reverse relationship. Respirable dust exposure mainly correlated with non-inflammasome related markers like CXCL8 and sST2.ConclusionsThe finding that NLRP3 inflammasome-related markers correlated with PM and quartz exposure suggest that this potent inflammatory cellular mechanism indeed is affected even at current exposure levels in Swedish iron foundries. The results highlight concerns regarding the safety of current exposure limits to respirable dust and quartz, and encourage continuous efforts to reduce exposure in dust and quartz exposed industries.

Thyroid cells from normal and autoimmune thyroid glands suppress T lymphocytes proliferation upon contact revealing a new regulatory inhibitory type of interaction independent of PD1/PDL1

Immune Checkpoint Receptors include a number of inhibitory receptors that limit tissue damage during immune responses; blocking PD-1/PD-L1 checkpoint receptor axis led to a paradigm shift in cancer immunotherapy but also to autoimmune adverse effects, prominently thyroid autoimmunity. Although PD-L1 is known to be expressed on thyroid follicular cells (TFCs) of autoimmune glands the role on PD-1/PD-L1 in the interaction between T cells and thyroid cells in the tissue has not been investigated. Here we report that autologous primary TFCs, but not transformed TFCs, inhibit CD4 and CD8 T cell proliferation but no cytokine production. This effect is not, however, mediated by PD-1/PD-L1 nor locally produced cytokines. Beta galactosidase analysis excluded culture-induced senescence as an explanation. High resolution flow cytometry demonstrated that autologous TFC/T cells co-culture induced the expansion of several clusters of double negative (DN) T cells characterized by high expression of activation markers and negative immune checkpoints. Single cell transcriptomic profiling demonstrated that dissociated TFC express numerous candidate molecules for mediating this suppressive activity, including CD40, E-Cadherin and TIGIT ligands. These ligands directly or through the generation of a suppressor population of DN T cells, and not the PD-1/PD-L1 axis, are most likely the responsible of TFC immunosuppressive activity. These results contribute to reveal the complex network of inhibitory mechanism that operate at the tissue level to restrain autoimmunity but also point to pathways, other that PD-1/PD-L1, that can contribute to tumor evasion.

Enhancing Efficacy of TCR-engineered CD4 + T Cells Via Coexpression of CD8α.

Adoptive cell therapy with T cells expressing affinity-enhanced T-cell receptors (TCRs) is a promising treatment for solid tumors. Efforts are ongoing to further engineer these T cells to increase the depth and durability of clinical responses and broaden efficacy toward additional indications. In the present study, we investigated one such approach: T cells were transduced with a lentiviral vector to coexpress an affinity-enhanced HLA class I-restricted TCR directed against MAGE-A4 alongside a CD8α coreceptor. We hypothesized that this approach would enhance CD4 + T-cell helper and effector functions, possibly leading to a more potent antitumor response. Activation of transduced CD4 + T cells was measured by detecting CD40 ligand expression on the surface and cytokine and chemokine secretion from CD4 + T cells and dendritic cells cultured with melanoma-associated antigen A4 + tumor cells. In addition, T-cell cytotoxic activity against 3-dimensional tumor spheroids was measured. Our data demonstrated that CD4 + T cells coexpressing the TCR and CD8α coreceptor displayed enhanced responses, including CD40 ligand expression, interferon-gamma secretion, and cytotoxic activity, along with improved dendritic cell activation. Therefore, our study supports the addition of the CD8α coreceptor to HLA class I-restricted TCR-engineered T cells to enhance CD4 + T-cell functions, which may potentially improve the depth and durability of antitumor responses in patients.

Rhesus macaque Bcl-6/Bcl-xL B cell immortalization: Discovery of HIV-1 neutralizing antibodies from lymph node

Many HIV-1 vaccines are designed to elicit neutralizing antibodies, and pre-clinical testing is often carried out in rhesus macaques (RMs). We have therefore adapted a method of B cell immortalization for use with RM B cells. In this system, RM B cells are activated with CD40 ligand and RM IL-21 before transduction with a retroviral vector encoding Bcl-6, Bcl-xL, and green fluorescent protein. Importantly, RM B cells from lymph nodes are more effectively immortalized by this method than B cells from PBMC, a difference not seen in humans. We suggest the discrepancy between these two tissues is due to increased expression of CD40 on RM lymph node B cells. Immortalized RM B cells expand long-term, undergo minimal somatic hypermutation, express surface B cell receptor, and secrete antibodies into culture. This allows for the identification of cells based on antigen specificity and/or functional assays. Here, we show the characterization of this system and its application for the isolation of HIV-1 neutralizing antibodies from a SHIV.CH505-infected animal, both with and without antigen probe. Taken together, we show that Bcl-6/xL immortalization is a valuable and flexible tool for antibody discovery in RMs, but with important distinctions from application of the system in human cells.

Platelet activation and coronavirus disease 2019 mortality: Insights from coagulopathy, antiplatelet therapy and inflammation

BackgroundCoronavirus disease 2019 (COVID-19) is associated with an inflammatory cytokine burst and a prothrombotic coagulopathy. Platelets may contribute to microthrombosis, and constitute a therapeutic target in COVID-19 therapy. AimTo assess if platelet activation influences mortality in COVID-19. MethodsWe explored two cohorts of patients with COVID-19. Cohort A included 208 ambulatory and hospitalized patients with varying clinical severities and non-COVID patients as controls, in whom plasma concentrations of the soluble platelet activation biomarkers CD40 ligand (sCD40L) and P-selectin (sP-sel) were quantified within the first 48hours following hospitalization. Cohort B was a multicentre cohort of 2878 patients initially admitted to a medical ward. In both cohorts, the primary outcome was in-hospital mortality. ResultsIn cohort A, median circulating concentrations of sCD40L and sP-sel were only increased in the 89 critical patients compared with non-COVID controls: sP-sel 40,059 (interquartile range 26,876–54,678)pg/mL; sCD40L 1914 (interquartile range 1410–2367)pg/mL (P<0.001 for both). A strong association existed between sP-sel concentration and in-hospital mortality (Kaplan-Meier log-rank P=0.004). However, in a Cox model considering biomarkers of immunothrombosis, sP-sel was no longer associated with mortality, in contrast to coagulopathy evaluated with D-dimer concentration (hazard ratio 4.86, 95% confidence interval 1.64–12.50). Moreover, in cohort B, a Cox model adjusted for co-morbidities suggested that prehospitalization antiplatelet agents had no significant impact on in-hospital mortality (hazard ratio 1.05, 95% CI 0.80–1.37; P=0.73). ConclusionsAlthough we observed an association between excessive biomarkers of platelet activation and in-hospital mortality, our findings rather suggest that coagulopathy is more central in driving disease progression, which may explain why prehospitalization antiplatelet drugs were not a protective factor against mortality in our multicentre cohort.

An mRNA mix redirects dendritic cells towards an antiviral program, inducing anticancer cytotoxic stem cell and central memory CD8+ T cells.

Dendritic cell (DC)-maturation stimuli determine the potency of these antigen-presenting cells and, therefore, the quality of the T-cell response. Here we describe that the maturation of DCs via TriMix mRNA, encoding CD40 ligand, a constitutively active variant of toll-like receptor 4 and the co-stimulatory molecule CD70, enables an antibacterial transcriptional program. Besides, we further show that the DCs are redirected into an antiviral transcriptional program when CD70 mRNA in TriMix is replaced with mRNA encoding interferon-gamma and a decoy interleukin-10 receptor alpha, forming a four-component mixture referred to as TetraMix mRNA. The resulting TetraMixDCs show a high potential to induce tumor antigen-specific T cells within bulk CD8+ T cells. Tumor-specific antigens (TSAs) are emerging and attractive targets for cancer immunotherapy. As T-cell receptors recognizing TSAs are predominantly present on naive CD8+ T cells (TN), we further addressed the activation of tumor antigen-specific T cells when CD8+ TN cells are stimulated by TriMixDCs or TetraMixDCs. In both conditions, the stimulation resulted in a shift from CD8+ TN cells into tumor antigen-specific stem cell-like memory, effector memory and central memory T cells with cytotoxic capacity. These findings suggest that TetraMix mRNA, and the antiviral maturation program it induces in DCs, triggers an antitumor immune reaction in cancer patients.

The CTLA‐4 immune checkpoint protein regulates PD‐L1:PD‐1 interaction via transendocytosis of its ligand CD80

CTLA‐4 and PD‐1 are key immune checkpoint receptors that are targeted in the treatment of cancer. A recently identified physical interaction between the respective ligands, CD80 and PD‐L1, has been shown to block PD‐L1/PD‐1 binding and to prevent PD‐L1 inhibitory functions. Since CTLA‐4 is known to capture and degrade its ligands via transendocytosis, we investigated the interplay between CD80 transendocytosis and CD80/PD‐L1 interaction. We find that transendocytosis of CD80 results in a time‐dependent recovery of PD‐L1 availability that correlates with CD80 removal. Moreover, CD80 transendocytosis is highly specific in that only CD80 is internalised, while its heterodimeric PD‐L1 partner remains on the plasma membrane of the antigen‐presenting cell (APC). CTLA‐4 interactions with CD80 do not appear to be inhibited by PD‐L1, but efficient removal of CD80 requires an intact CTLA‐4 cytoplasmic domain, distinguishing this process from more general trogocytosis and simple CTLA‐4 binding to CD80/PD‐L1 complexes. These data are consistent with CTLA‐4 acting as modulator of PD‐L1:PD‐1 interactions via control of CD80.

An open-label, multicenter study investigating RP3 oncolytic immunotherapy in combination with first- or second-line systemic atezolizumab and bevacizumab therapy in patients with locally advanced unresectable or metastatic hepatocellular carcinoma.

TPS623 Background: Despite advances in treatment for unresectable hepatocellular carcinoma (HCC), long-term survival rates remain poor. The combination of bevacizumab (Bev) and atezolizumab (Atezo) is the preferred frontline therapy for advanced HCC, but a minority of patients (pts) respond, and secondary resistance usually occurs within months. HCC has an immune-suppressed tumor microenvironment, mediated by activated immune checkpoint signaling and angiogenesis pathways, which may contribute to therapeutic resistance. RP3 is a genetically modified herpes simplex virus 1 (HSV-1) that expresses the fusogenic GALV-GP R- protein, an anti–CTLA-4 antibody-like molecule, CD40 ligand, and 4-1BB ligand. The direct oncolytic effect coupled with immune stimulation by RP3 in the tumor microenvironment is intended to provide systemic anti-tumor activity and reverse therapeutic resistance to anti-PD-1/PD-L1 agents. Pre-clinical data has demonstrated improved distribution of oncolytic HSV within tumors in combination with Bev supporting the clinical combination of RP3 with Bev. RP3 combined with anti-PD1 therapy has demonstrated clinical activity and safety in pts with various malignant solid tumors. This study will evaluate the safety and efficacy of RP3 combined with Atezo and Bev as 1st (1L) and 2nd (2L) line systemic therapies for inoperable and advanced HCC. Methods: The 1L and 2L cohorts will each enroll up to 30 pts with advanced, unresectable HCC. Pts in the 1L cohort may not have received prior systemic treatment; pts in the 2L cohort must have progressed on or following one prior line of systemic therapy, which must have included a PD-(L)1 directed agent. Key inclusion criteria include advanced unresectable HCC with at least 1 measurable tumor of ≥1 cm in longest diameter, Childs-Pugh Class A, and ECOG Score of 0 or 1. Important exclusion criteria include untreated esophageal and/or gastric varices with bleeding or at high risk for bleeding, and macroscopic invasion of tumor into major blood vessels and/or main bile ducts. Pts with a history of medically refractory hepatic encephalopathy and/or hepato-renal syndrome are also excluded. Pts in 1L cohort will receive 1200 mg Atezo and 15mg/kg Bev Q3W together with RP3 intratumorally (IT) Q3W for a total of up to 8 doses. In 2L pts will receive RP3 Q2W for 4 doses with Bev Q3W starting on C1D1, then RP3 and Bev Q3W for up to 4 more doses with Atezo Q3W being added on C4D1. The primary endpoint includes overall response rate (ORR) by RECIST v1.1. Secondary endpoints are safety, ORR using HCC mRECIST, duration of response, complete response rate, and progression-free survival.