Degranulation Assay Research Articles

Glyco-binding domain chimeric antigen receptors as a new option for cancer immunotherapy

In the last decade, treatment using Chimeric Antigen Receptor (CAR) are largely studied and demonstrate the potential of immunotherapeutic strategies, as seen mainly for blood related cancers. Still, efficient CAR-T cell approaches especially for the treatment of solid tumors are needed. Tn- and Sialyl-Tn antigens are tumor associated carbohydrate antigens correlating with poor prognosis and tumor metastasis on a variety of tumor entities. These glycans can be recognized by CD301 (CLEC10A, MGL), which is a surface receptor found primarily on immune cells. In the present study, we hypothesized, that it is possible to use newly generated CD301-bearing CARs, enabling cytotoxic effector cells to recognize and eliminate breast cancer cells. Thus, we genetically modified human NK92 cells with different chimeric receptors based on the carbohydrate recognition domain (CRD) of human CD301. We assessed their cytotoxic activity in vitro demonstrating the specific recognition of CD301 ligand positive cell lines. These results were confirmed by degranulation assays and in cytokine release assays. Overall, this study demonstrates CD301-CARs represent a cost-effective and fast alternative to conventional scFv CARs for cancer immunotherapy.

Molecular Cloning and Characterization of a Novel Antimicrobial Peptide from the Skin of Kaloula pulchra.

Bacterial resistance to all currently available conventional antibiotics has caused a global public health crisis and led to an imperative search for new agents. Antimicrobial peptides (AMPs) are essential components of host innate immune defense against microbial invasions. The objective of this study was to report a novel AMP, brevinin-2KP, from the skin of the black Kaloula pulchra frog and describe its structural and biological characterization. The physical and chemical parameters of brevinin-2KP were predicted with the ExPASy Bioinformatics Resource Portal. The assembled sequences were aligned with ClustalW, and the phylogenetic tree was constructed using Mega. Circular dichroism (CD) experiments were carried out to identify the secondary structure and the stability of peptide in different solvent environments. The cytotoxicity of brevinin-2KP was evaluated by the MTT test. To determine antibacterial activity of brevinin- 2KP, a standard two-fold broth dilution method was used. SEM was carried out to observe the morphological change in the bacterial treated by brevinin-2KP. The live/dead bacterial viability was measured with a LIVE/DEAD® BacLight kit. Histamine release and mast cell degranulation assays were performed. The precursor of brevinin-2KP contains 72 amino acid residues, including a conserved signal peptide, acidic propeptide with KR residues, and mature peptide with a sequence of GVITDALKGAAKTVAAELLKKAHCKLTNSC. Phylogenetic analysis based on the amino acid sequences of 34 brevinin-2 peptides from 30 anuran species demonstrates that K. pulchra is genetically closely related to the genus Hylarana. The CD spectra analysis indicates that brevinin-2KP adopts random coil in the water and an organized α-helical conformation in SDS solution. Further, this secondary structure is stable under high salt and high-temperature conditions. Brevinin-2KP is weakly active towards the tested Gram-positive and Gram-negative bacteria as well as fungi due to its membranolytic action. Moreover, brevinin-2KP inhibits the proliferation of several mammal cells with IC50 values ranging from 3.27 to 59.75 μM. In addition, brevinin-2KP promotes degranulation and histamine release of mast cells, indicating that it is involved in the inflammatory response. This is the first report on AMP identified from the skin of K. pulchra. Brevinin-2KP adopts a typical amphipathic α-helix conformation in membrane mimic environment and shows antimicrobial and antitumor activities by potential membranolytic mechanism. In addition, brevinin-2KP can promote degranulation and histamine release of mast cells. Brevinin-2KP is expected to become a good drug temple molecule.

Biological screening of a unique drug library targeting MRGPRX2.

Allergic drug reaction or drug allergy is an immunologically mediated drug hypersensitivity reaction (DHR). G-protein coupled receptors (GPCRs) are common drug targets and communicate extracellular signals that initiate cellular responses. Recent evidence shows that GPCR MRGPRX2 is of major importance in IgE-independent pseudo-allergic DHRs based on the suspected interactions between many FDA-approved peptidergic compounds and MRGPRX2. Our aim was to uncover novel MRGPRX2-selective and -potent agonists as drug candidates responsible for clinical features of pseudo-allergic DHRs. We conducted a primary high-throughput screening (HTS), coupled with mutagenesis targeting the MRGPRX2 N62S mutation, on a panel of 3,456 library compounds. We discovered pharmacologically active hit compounds as agonists of the MRGPRX2 protein according to high degrees of potency evaluated by the calcium response and validated by the degranulation assay. Using the molecular tool Forge, we also characterized the structure-activity relationship shared by identified hit compounds. The alternative allele of single nucleotide polymorphism rs10833049 (N62S) in MRGPRX2 demonstrated loss-of-function property in response to substance P and antineoplastic agent daunorubicin hydrochloride. We applied a unique assay system targeting the N62S mutation to the HTS and identified 84 MRGPRX2-selective active hit compounds representing diverse classes according to primary drug indications. The top five highly represented groups included fluoroquinolone and non-fluoroquinolone antibiotics; antidepressive/antipsychotic; antihistaminic and antineoplastic agents. We classified hit compounds into 14 clusters representing a variety of chemical and drug classes beyond those reported, such as opioids, neuromuscular blocking agents, and fluoroquinolones. We further demonstrated MRGPRX2-dependent degranulation in the human mast cell line LAD2 cells induced by three novel agonists representing the non-fluoroquinolone antibiotics (bacitracin A), anti-allergic agents (brompheniramine maleate) and tyrosine-kinase inhibitors (imatinib mesylate). Our findings could facilitate the development of interventions for personalized prevention and treatment of DHRs, as well as future pharmacogenetic investigations of MRGPRX2 in relevant disease cohorts.

INFLAMMATORY CYTOKINES PERTURB NK CELL FUNCTION AND GENE SIGNATURE IN CHRONIC MYELOID LEUKEMIA

Natural killer (NK) cells have the capacity to eliminate malignant cells by releasing cytotoxic granules, which makes them a potent anti-cancer immunotherapeutic. NK cells'mature phenotype and counts positively correlate with prolonged treatment-free survival in chronic myeloid leukemia (CML), while dysfunctional NK cells are predictive of relapse in CML patients. The molecular drivers of this impairment during leukemic progression remain unclear. Given the established role of inflammatory cytokines (namely IL1β, IL-6, GM-CSF, TNFα) in driving myeloid malignancies, we aimed to define their effect on NK cell phenotype and function using a unique pre-clinical chimeric mouse model of CML. We hypothesize that leukemic cytokines impede NK cell maturation and cytotoxicity and shift NK cells toward a pro-tumor cytokine-producing phenotype. We establish a chimeric mouse model of CML by transplanting bone marrow BCR-ABL+ CD45.2 cells into sub-lethally irradiated CD45.1 C57BL/6 wild-type mice. For controls, mice are transplanted with non-mutant cells. Such chimeras represent a robust model to study the exposure of non-transformed host cells to leukemia. At the endpoint, splenic, blood, and bone marrow NK cells are harvested and profiled by flow cytometry, RT-qPCR, scRNA-seq, or used for degranulation assay. Blood samples are collected to obtain serum. Consistent with clinical observations, NK cells are severely reduced in numbers and frequencies and display an immature phenotype and diminished expression of activating receptors (Nkp46, Ly49D) while upregulating inhibitory molecules (Lag-3, TIGIT, NKG2A) during leukemia. Moreover, CML-exposed NK cells have a higher proliferation rate and a greater cell death indicating perturbed homeostasis. We revealed a preferential loss of terminally mature CD27−CD11b+ NK subset in CML mice, suggesting a functional shift from cytotoxic phenotype toward the immature cytokine-producing. In agreement, CML-exposed NK cells show impaired cytotoxicity as measured by target-specific degranulation ex vivo . Next, we mapped the transcriptional landscape of CML-exposed NK cells using single-cell RNA-sequencing. We confirmed decreased gene expression of NK maturation and cytotoxicity markers ( Itgam, Cx3cr1, Prf1, Gzma ) and upregulation of inhibitory molecules ( Tigit, Lag-3, Pdcd1lg1/2) and proteins involved in the cell cycle and senescence ( Mki67, Ccna, Ccnb ). Importantly, CML-exposed NK cells had significantly increased mRNA levels of inflammatory cytokines including IL1β, TNF, GM-CSF; cytokine receptors Il6st, Il-2Ra; and negative regulators of STAT signaling SOCS1/2/3 and CIS. Furthermore, IL-6-STAT3 and IL1β/TNF-signaling gene signatures were enriched in CML-exposed NK cells – an effect that is likely to be triggered by inflammatory cytokines. Thus, we next found that peripheral blood serum from CML mice dampens healthy NK cell degranulation ex vivo , compared to serum from control mice. Further ELISA analysis revealed elevated levels of IL-6, IL-1, TNF, and GM-CSF in CML serum samples. Some of these cytokines might be secreted by functionally skewed NK cells, as suggested by gene expression analysis. Altogether, our data suggest that leukemic cytokines contribute to NK cell dysfunction and polarize them toward a pro-inflammatory phenotype, representing optimal targets for NK-boosting CML immunotherapies.

Discovery and Development of First-in-Class ACKR3/CXCR7 Superagonists for Platelet Degranulation Modulation.

The atypical chemokine receptor 3 (ACKR3), formerly known as CXC-chemokine receptor 7 (CXCR7), has been postulated to regulate platelet function and thrombus formation. Herein, we report the discovery and development of first-in-class ACKR3 agonists, which demonstrated superagonistic properties with Emax values of up to 160% compared to the endogenous reference ligand CXCL12 in a β-arrestin recruitment assay. Initial in silico screening using an ACKR3 homology model identified two hits, C10 (EC50 19.1 μM) and C11 (EC50 = 11.4 μM). Based on these hits, extensive structure-activity relationship studies were conducted by synthesis and testing of derivatives. It resulted in the identification of the novel thiadiazolopyrimidinone-based compounds 26 (LN5972, EC50 = 3.4 μM) and 27 (LN6023, EC50 = 3.5 μM). These compounds are selective for ACKR3 versus CXCR4 and show metabolic stability. In a platelet degranulation assay, these agonists effectively reduced P-selectin expression by up to 97%, suggesting potential candidates for the treatment of platelet-mediated thrombosis.

Marek's disease virus-specific T cells proliferate, express antiviral cytokines but have impaired degranulation response.

The major histocompatibility complex (MHC) haplotype is one of the major determinants of genetic resistance and susceptibility of chickens to Marek’s disease (MD) which is caused by an oncogenic herpesvirus; Marek’s disease virus (MDV). To determine differential functional abilities of T cells associated with resistance and susceptibility to MD, we identified immunodominant CD4+TCRvβ1 T cell epitopes within the pp38 antigen of MDV in B19 and B21 MHC haplotype chickens using an ex vivo ELISPOT assay for chicken IFN-gamma. These novel pp38 peptides were used to characterize differential functional abilities of T cells as associated with resistance and susceptibility to MD. The results demonstrated an upregulation of cytokines (IL-2, IL-4, IL-10) and lymphocyte lysis-related genes (perforin and granzyme B) in an antigen specific manner using RT-PCR. In the MD-resistant chickens (B21 MHC haplotype), antigen-specific and non-specific response was highly skewed towards Th2 response as defined by higher levels of IL-4 expression as well as lymphocyte lysis-related genes compared to that in the MD-susceptible chicken line (B19 MHC haplotype). Using CD107a degranulation assay, the results showed that MDV infection impairs cytotoxic function of T cells regardless of their genetic background. Taken together, the data demonstrate an association between type of T cell response to pp38 and resistance to the disease and will shed light on our understanding of immune response to this oncogenic herpesvirus and failure to induce sterile immunity.

Protease inhibitor from Libidibia ferrea seeds attenuates inflammatory and nociceptive responses in mice

Ethnopharmacological relevanceLibidibia ferrea (Mart. ex. Tul.) L.P. Queiroz is a Brazilian native tree locally known as jucá and pau-ferro, and it has been used in folk medicine for relieving, asthma, bronchitis, sore throat, rheumatism, enterocolitis and fever. The anti-inflammatory properties of L. ferrea were confirmed for its stem, fruit, leaves, bark and seeds extracts, however little is known about the natural compounds that may be associated with that response. Aim of this studyIn a normal physiological condition, many enzymes play an important role in catalyzing biological functions. Among them, proteases are of great interest. Although they take part of many biological systems, as the inflammatory process, when deregulated, proteases may cause system malfunctions, such as under- or overproduction of cytokines, or immune cells activation. Thus, protease inhibitors prevent these immune responses by regulating proteases. The objective of this study was to evaluate the anti-inflammatory and anti-nociceptive response of a protease inhibitor purified from L. ferrea seeds (LfTI). Materials and methodsIn vitro (5, 50 and 250 μg/mL of LfTI) and in vivo (0.6, 3 e 15 mg/kg of LfTI) assays were performed. Male Swiss mice weighing 18–25 g were used for cell harvesting and for the in vivo assays. The anti-inflammatory activity was analyzed in vitro by macrophage cytotoxicity, hydrogen peroxide (H2O2) production, and cell adhesion assays; and in vivo by leukocyte recruitment, nitric oxide (NO) production, vascular permeability, paw edema and mast cell degranulation assays. The anti-nociceptive activity was evaluated through abdominal writhing test induced by acetic acid and formalin sensitization. ResultsOur results showed that, in vitro, LfTI is not cytotoxic. Also, LfTI (50 μg/mL) inhibited macrophage H2O2 production (48.2%), and adhesion (48.4%). LfTI (0.6, 3 e 15 mg/kg) decreased polymorphonuclear cell recruitment dose-dependently, and it inhibited NO production (53%), vascular permeability (40.7%) and paw edema at 3 mg/kg at different time, but it did not inhibit mast cell degranulation. Besides, LfTI did not inhibit either the number of writhing or the licking time in the formalin test in the second phase (inflammatory). However, LfTI (3 mg/kg) inhibited licking time at the first phase (neurogenic) in the formalin sensitization (46.1%). ConclusionsOur results show that LfTI has anti-inflammatory and antinociceptive (neurogenic pain) effects, and these effects might be associated with the inhibition of inflammatory proteases and/or protease-activated receptors activation hindering.

Anemoside B4 protects against IgE-dependent allergic responses by suppressing the PLC/IP3 and JAK/STAT3 pathways

Anemoside B4 (AB4) is a natural triterpenoid abundant in the roots of Pulsatilla chinensis. Although various biological activities have been widely attributed to AB4, few studies have focused on its antiallergic effects. In this study the inhibitory effects of AB4 on immunoglobulin E (IgE)-mediated allergic responses were investigated, both in vitro and in vivo, and the mechanism of its effects. IgE-mediated passive cutaneous anaphylaxis was used to elucidate the antiallergic effects of AB4 in vivo. The degranulation assay, calcium imaging, and cytokine and chemokine release in the laboratory of allergic disease 2 (LAD2) cell line were used to evaluate the antiallergic effect of AB4 in vitro. Pathological staining was performed to analyze angiectasis. Western blot analysis was performed to investigate the downstream signaling pathways. AB4 dose-dependently attenuated ovalbumin/IgE-induced paw swelling in mice, and reduced the serum concentrations of tumor necrosis factor-alpha and C–C motif chemokine 2. In addition, AB4 suppressed IgE-mediated LAD2 cell degranulation, calcium influx, and PLC/IP3 and JAK/STAT3 phosphorylation. Our results suggest that AB4 inhibits allergic reactions through the PLC/IP3 and JAK/STAT3 pathways.

Practice patterns and outcomes of hemophagocytic lymphohistiocytosis in adults: a 2-decade provincial retrospective review.

Hemophagocytic lymphohistiocytosis (HLH) is a heterogeneous, life-threatening clinical syndrome. There are scarce data on the quality of care in HLH or data comparing treatment patterns and outcomes between different triggers. We aimed to examine quality-of-care indicators and outcomes in adults with various HLH triggers. In this multi-centre retrospective cohort study of adult HLH in the province of Alberta, Canada (1999-2019), we examined quality indicators including diagnostic testing, time to diagnosis and treatment and trigger identification. We also compared treatment regimens and outcomes across HLH triggers. Logistic regression was used to identify predictors of etoposide use. Overall survival (OS) was estimated using the Kaplan-Meier method. We identified 97 patients; 66 (68%) were male. Triggers included malignancy (36%), infection (35%), autoimmune disease (21%) and idiopathic/others (8%). Specialized tests such as sCD25 (53%) and natural killer degranulation assay (19%) were under-performed, as were testing for infectious triggers. Etoposide was administered in only 33 (34%). Neutropenia, hyperbilirubinemia and hyperferritinemia, but not age, sex and comorbidities, were significant predictors of etoposide use. At median follow-up of 32months, median OS was 18.8months. Worse OS was seen in malignancy-associated and idiopathic HLH (log-rank P < 0.001). Our study showed low rates of specialized testing such as sCD25 and a low rate of etoposide use. Development of a standardized provincial protocol has the potential to improve quality of care in adult HLH.

Salicylaldehyde Suppresses IgE-Mediated Activation of Mast Cells and Ameliorates Anaphylaxis in Mice.

Mast cells (MCs) play key roles in IgE-mediated immunoresponses, including in the protection against parasitic infections and the onset and/or symptoms of allergic diseases. IgE-mediated activation induces MCs to release mediators, including histamine and leukotriene, as an early response, and to produce cytokines as a late phase response. Attempts have been made to identify novel antiallergic compounds from natural materials such as Chinese medicines and food ingredients. We herein screened approximately 60 compounds and identified salicylaldehyde, an aromatic aldehyde isolated from plant essential oils, as an inhibitor of the IgE-mediated activation of MCs. A degranulation assay, flow cytometric analyses, and enzyme-linked immunosorbent assays revealed that salicylaldehyde inhibited the IgE-mediated degranulation and cytokine expression of bone-marrow-derived MCs (BMMCs). The salicylaldehyde treatment reduced the surface expression level of FcεRI, the high affinity receptor for IgE, on BMMCs, and suppressed the IgE-induced phosphorylation of tyrosine residues in intercellular proteins, possibly Lyn, Syk, and Fyn, in BMMCs. We also examined the effects of salicylaldehyde in vivo using passive anaphylaxis mouse models and found that salicylaldehyde administration significantly enhanced the recovery of a reduced body temperature due to systemic anaphylaxis and markedly suppressed ear swelling, footpad swelling, and vascular permeability in cutaneous anaphylaxis.

The effect of substance P and its common in vivo-formed metabolites on MRGPRX2 and human mast cell activation.

The tachykinin neuropeptide substance P (SP) is the canonical agonist peptide for the neurokinin 1 receptor (NK1R). More recently, it has also been shown to activate the Mas‐related G protein‐coupled receptor X2 (MRGPRX2) receptor on mast cells (MCs), triggering degranulation and release of inflammatory mediators. SP undergoes rapid C‐terminal truncation in vivo by a number of proteases to generate the metabolites SP(1–9)‐COOH and in particular SP(1–7)‐COOH. While the C terminus of SP is critical for NK1R activation, studies have shown that the peptide polycationic N terminus is key for MRGPRX2 and mast cell activation. The study thus aimed to determine if the C‐terminally truncated metabolites of SP, SP(1–9)‐COOH, and SP(1–7)‐COOH retained stimulatory activity at MRGPRX2. SP, SP(1–9)‐COOH, and SP(1–7)‐COOH were synthesized and tested on HEK293 cells expressing NK1R or MRGPRX2, and LAD2 human mast cells, to determine the activity of SP and its metabolites in Ca2+ mobilization, degranulation, and cytokine assays. As expected from prior studies, both C‐terminally truncated SP metabolites had essentially no activity at NK1R, even at very high concentrations. In contrast, the in vivo metabolite of SP, SP(1–9)‐COOH retained ability to activate MRGPRX2 across all parameters tested, albeit with reduced potency compared to intact SP. SP(1–7)‐COOH did not produce any significant MRGRPX2 activation. Our results suggest that the SP metabolite, SP(1–9)‐COOH, may play a regulatory role through the activation of MRGPRX2. However, given the relatively low potency of both SP and SP(1–9)‐COOH at MRGPRX2, additional work is needed to better understand the biological importance of this expanded SP/MRGPRX2 pathway.

Computational prediction and experimental analysis of the nanoparticle-protein corona: Showcasing an in vitro-in silico workflow providing FAIR data

Extensive investigation and characterisation of nanoparticle-protein conjugates are imperative to assess potential nanoparticle-induced hazards for humans and the environment, predict adverse biological effects, and identify suitable nanoparticles for medical applications. Investigating the formation of the nanoparticle protein corona solely based on experimental analysis is currently very time-consuming and cost-intensive. Therefore, development of prediction tools based on in silico modelling is much-needed in order to provide viable alternative approaches and accelerate nanomaterial risk assessment at the early development stage. This work aimed to validate currently emerging in silico protein corona modelling tools with experimental results and to reveal the models’ potentials and limitations thereby contributing to the improvement of their predictive power. Comprehensive data and metadata sets of the obtained in vitro and in silico results were collected and annotated in the NanoCommons Knowledge Base to facilitate data Findability, Accessibility, Interoperability, and Reusability (FAIRness) in nanosafety assessment. In silico protein corona predictions (in silico modelling with UnitedAtom) and in vitro investigation of corona formation (binding and selectivity studies with eight different proteins, mixtures thereof, and an allergenic effector cell degranulation assay) on differently coated SiO2 nanoparticles were aligned and the results, in the first run, revealed substantial deviations. Therefore, we attempted to identify the potential and limitations in the modelling and provided recommendations to improve the model. Similar iteractive approaches, as described here, based on the verification versus rebuttal of data from in silico procedures by in vitro analyses, complemented by comprehensive data and metadata collection according to the FAIR principles, are expected to help optimise future prediction certainties and improve in silico modelling.

Combined Blockade Of PD-1 and TIGIT is not Sufficient to Improve the Function Of CD8+ T-Cells in Chronic Lymphocytic Leukemia.

Blockade of immune checkpoint receptors in the treatment of cancers has been mentioned in several studies. Here, we investigated the efficacy of combined blockade of two inhibitory receptors, PD-1 and TIGIT, in restoring functional features of CD8+ T-cells in CLL. CD8+ T-cells were separated from the peripheral blood of 11 CLL patients and targeted with malignant B-cells isolated from the same patients. Cells were then stimulated with anti-CD3/CD28 and PMA/ionomycin to assess their proliferative response and cytotoxic activity using MTT and CD107a degranulation assays, respectively. Cytokine production of isolated CD8+ T-cells was also determined using ELISA. There were no significant differences in proliferation and cytotoxic activity of CD8+ T-cells co-blocked with anti-PD-1/TIGIT compared to those single blocked with anti-PD-1, anti-TIGIT, or the control antibody. There was no significant difference in cytokine production of mentioned groups, either. Collectively, combined blockade of PD-1 and TIGIT failed to restore the proliferation and function of CD8+ T-cells isolated from CLL patients.

Assessment of the safety of the cationic arginine-rich peptides (CARPs) poly-arginine-18 (R18 and R18D) in ex vivo models of mast cell degranulation and red blood cell hemolysis

Our laboratory focuses on the development of novel neuroprotective cationic peptides, such poly-arginine-18 (R18: 18-mer of l-arginine; net charge +18) and its d-enantiomer R18D in stroke and other brain injuries. In the clinical development of R18/R18D, their cationic property raises potential safety concerns on their non-specific effects to induce mast cell degranulation and hemolysis. To address this, we first utilised primary human cultured mast cells (HCMCs) to examine anaphylactoid effects. We also included as controls, the well-characterised neuroprotective TAT-NR2B9c peptide and the widely used heparin reversal peptide, protamine. Degranulation assay based on β-hexosaminidase release demonstrated that R18 and R18D did not induce significant mast cell degranulation in both untreated (naïve) and IgE-sensitised HCMCs in a dose-response study to a maximum peptide concentration of 16 μM. Similarly, TAT-NR2B9c and protamine did not induce significant mast cell degranulation. To examine hemolytic effects, red blood cells (RBCs), were incubated with the peptides at a concentration range of 1–16 μM in the absence or presence of 2% plasma. Measurement of hemoglobin absorbance revealed that only R18 induced a modest, but significant degree of hemolysis at the 16 μM concentration, and only in the absence of plasma. This study addressed the potential safety concern of the application of the cationic neuroprotective peptides, especially, R18D, on anaphylactoid responses and hemolysis. The findings indicate that R18, R18D, TAT-NR2B9c and protamine are unlikely to induce histamine mediated anaphylactoid reactions or RBC hemolysis when administered intravenously to patients.

Abstract 287: Engineered 3D tissues facilitate preclinical immunotherapy studies in fully human platforms

Abstract Cancer treatment has evolved with the advent of immunotherapies. This drug class now plays a key role in supplementing or even replacing chemotherapies as first line treatments. Immunotherapies such as immune checkpoint inhibitors, bispecific antibodies, and chimeric antigen receptor T-cells (CAR-T) are just a few of the therapeutic strategies currently utilized to promote an anti-cancer response. Preclinical testing of current and novel immune modulators is necessary to expand our knowledge of these drugs and better predict their efficacy. Animal models have numerous deficiencies and complications that make them insufficient for accurate representation of human response, including an insufficient human immune system and a lack of genomic heterogeneity. Thus, there is an urgent need to develop preclinical models that incorporate human tumors and autologous immune cells together to create an accurate microenvironment for effective monitoring of tumor and immune cell interactions. We have developed a tumor-type agnostic platform in which 3D cell cultures are engineered with cells from resected human tumors. This ex vivo model not only recapitulates the tumor immune microenvironment (TIME), but it includes multiple downstream read-outs to address a single question such as whether an agent or combination of agents stimulates an immune response resulting in an anti-tumor effect. Our model can incorporate tumor and immune cells directly from the TIME or tumor cells from patient-derived xenografts or organoids which are then supplemented with allogenic or autologous immune cells or even CAR-T cells. Immune response can be detected via T-cell activation, degranulation assays, and immune cell-mediated tumor cell killing which can be multiplexed with anti-tumor activity assays. We can measure a reduction in viable tumor cells via flow cytometry following treatment or at increasing effector to target cell ratios, detect fluctuations in production and secretion of granzyme B indicating T-cell cytotoxic activity, and evaluate reduced cell viability for synergy assessments of drug combinations. We have detected increased secretion of IFNγ with combination drug treatment compared to single agent treatment. Macrophage polarization can be monitored with co-culture of CD14+ cells with tumor cells in our system inducing an M2 phenotype from an M1 phenotype. Finally, we can detect dendritic cell maturation following stimulation by measuring increased expression of MHC class II and CD103. Taken together this model system supports complex cell-cell interactions necessary to detect immunotherapy response and represents an ideal preclinical testing platform. Citation Format: Kathryn M. Appleton, Katy A. Lassahn, Ashley K. Elrod, Teresa M. DesRochers. Engineered 3D tissues facilitate preclinical immunotherapy studies in fully human platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 287.

Leukemia derived dendritic cell (DCleu) mediated immune response goes along with reduced (leukemia-specific) regulatory T-cells

The blastmodulatory Kit-M, composed of granulocyte-macrophage colony-stimulating-factor (GM-CSF) and Prostaglandin E1 (PGE1), is known to convert myeloid leukaemic blasts (from AML patients) into leukaemia derived dendritic cells (DCleu), which activate immunoreactive cells to gain antileukemic/leukaemia-specific activity. In this study we had a special focus on the influence of Kit-M treated, DC/DCleu containing patients’whole blood (WB, n = 16) on the provision of immunosuppressive regulatory T-cells.We could confirm that Kit-M significantly increased frequencies of (mature) dendritic cells (DC) and DCleu from leukemic whole blood (WB) without induction of blast proliferation. After mixed lymphocyte culture (MLC) with patients’ T-cells we confirmed that DCleu mediated leukemia-specific responses- going along with activated and leukemia-specific T- and NK-cells in an intracellular cytokine staining assay (ICS) and a degranulation assay (Deg)- resulted in an increased anti-leukemic cytotoxicity (Cytotoxicity Fluorolysis Assay = CTX). We could demonstrate that (leukemia-specific) CD4+ and CD8+ regulatory T-cell population (Treg) decreased significantly after MLC compared to controls. We found significant positive correlations of leukemia-specific CD3+CD4+ cells with frequencies of (mature) DCleu. Achieved anti-leukemic cytotoxicity correlated significantly positive with leukemia-specific CD3+CD8+ cells and significantly negatively with (leukemia-specific) Treg.In summary we demonstrate that immunesuppressive (leukemia-specific) regulatory T-cells are significantly downregulated after Kit-M triggered MLC- going along with a (reinstalled) antileukemic reactivity of the immune system (as demonstrated with functional assays ICS, Deg, CTX).

Targeting CD123 in blastic plasmacytoid dendritic cell neoplasm using allogeneic anti-CD123 CAR T cells

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with poor outcomes with conventional therapy. Nearly 100% of BPDCNs overexpress interleukin 3 receptor subunit alpha (CD123). Given that CD123 is differentially expressed on the surface of BPDCN cells, it has emerged as an attractive therapeutic target. UCART123 is an investigational product consisting of allogeneic T cells expressing an anti-CD123 chimeric antigen receptor (CAR), edited with TALEN® nucleases. In this study, we examine the antitumor activity of UCART123 in preclinical models of BPDCN. We report that UCART123 have selective antitumor activity against CD123-positive primary BPDCN samples (while sparing normal hematopoietic progenitor cells) in the in vitro cytotoxicity and T cell degranulation assays; supported by the increased secretion of IFNγ by UCART123 cells when cultured in the presence of BPDCN cells. UCART123 eradicate BPDCN and result in long-term disease-free survival in a subset of primary patient-derived BPDCN xenograft mouse models. One potential challenge of CD123 targeting therapies is the loss of CD123 antigen through diverse genetic mechanisms, an event observed in one of three BPDCN PDX studied. In summary, these results provide a preclinical proof-of-principle that allogeneic UCART123 cells have potent anti-BPDCN activity.

LC-MS/MS metabolomics-facilitated identification of the active compounds responsible for anti-allergic activity of the ethanol extract of Xenostegia tridentata.

In vivo and in vitro anti-allergic activities of ethanol extract of Xenostegia tridentata (L.) D.F. Austin & Staples were investigated using passive cutaneous anaphylaxis reaction assay and RBL-2H3 cell degranulation assay, respectively. The crude ethanol extract exhibited promising activities when compared with the known anti-allergic agents, namely dexamethasone and ketotifen fumarate. The ethyl acetate subfraction showed the highest anti-allergic activity among various sub-partitions and showed better activity than the crude extract, consistent with the high abundance of total phenolic and flavonoid contents in this subfraction. LC-MS/MS metabolomics analysis and bioassay-guided isolation were then used to identify chemical constituents responsible for the anti-allergic activity. The results showed that major components of the ethyl acetate subfraction consist of 3,5-dicaffeoylquinic acid, quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside and luteolin-7-O-glucoside. The inhibitory activity of the isolated compounds against mast cell degranulation was validated, ensuring their important roles in the anti-allergic activity of the plant. Notably, besides showing the anti-allergic activity of X. tridentata, this work highlights the role of metabolomic analysis in identifying and selectively isolating active metabolites from plants.

Dok-1 regulates mast cell degranulation negatively through inhibiting calcium-dependent F-actin disassembly

In food allergies, antigen-induced aggregation of FcεRI on mast cells initiates highly ordered and sequential signaling events. Dok-1(downstream of tyrosine kinase 1), undergoes intense tyrosine phosphorylation upon FcεRI stimulation, which negatively regulates Ras/Erk signaling and the subsequent cytokine release, but it remains unclear whether Dok-1 regulates Fc-mediated degranulation. In this study, we investigated the role of Dok-1 in FcεRI-mediated degranulation. Dok-1 overexpressing RBL-2H3 cells were established. Degranulation, immunoprecipitation, co-immunoprecipitation, immunoblotting and flow cytometry assay were performed to explore the effects of Dok-1 and its underlying mechanisms. We found that, following FcεRI activation, Dok-1 was recruited to the plasma membrane, leading to tyrosine phosphorylation. Phosphorylated Dok-1 inhibits FcεRI-operated calcium influx, and negatively regulated degranulation by inhibiting calcium-dependent disassembly of actin filaments. Our data revealed that Dok-1 is a negative regulator of FcεRI-mediated mast cell degranulation. These findings contribute to the identification of therapeutic targets for food allergies.

Determination of immunogenic epitopes in major house dust mite allergen, Der p 2, via nanoallergens

BackgroundDespite the high prevalence of allergic asthma, currently, avoidance of the responsible allergens, which is nearly impossible for allergens such as house dust mite (HDM), remains among the most effective treatment. Consequently, determination of the immunogenic epitopes of allergens will aid in developing a better understanding of the condition for diagnostic and therapeutic purposes. Current methods of epitope identification, however, only evaluate immunoglobulin E-epitope binding interactions, which is not directly related to epitope immunogenicity. ObjectiveTo determine and rank the immunogenicity of the epitopes of major HDM allergen, Der p 2. MethodsWe performed degranulation assays with RBL-SX38 cells primed using patient plasma and challenged with nanoallergens which multivalently displayed epitopes to study the relative immunogenicity of various epitopes of Der p 2. Nanoallergens were used to evaluate epitopes individually or in combination. ResultsWhen evaluated using 3 patient samples, 3 epitopes in 2 distal regions of Der p 2 were identified as highly immunogenic when presented in combination, whereas no individual epitope triggered relevant degranulation. One of the epitopes (69-DPNACHYMKCPLVKGQQY-86) was identified to be cooperatively immunogenic when combined with other epitopes. ConclusionOur study highlights the importance of conformational epitopes in HDM-related allergies. This study also provides further evidence of the versatility of nanoallergens and their value for functional characterization of allergy epitopes, by ranking the Der p 2 epitopes according to immunogenicity. We believe that nanoallergens, by aiding in identification and understanding of immunogenic epitopes, will provide a better understanding of the manifestation of the allergic condition and potentially aid in developing new treatments.