Cell Compartment Research Articles

Genome-wide characterization of melatonin biosynthetic pathway genes in carnation (Dianthus caryophyllus L.) and their expression analysis in response to exogenous melatonin

Carnation (Dianthus caryophyllus L.) is a prominent floricultural crop valued for its diverse colors, offering significant economic and ornamental value globally. However, the global demand for its flowers makes flower yield an important attribute that relies on the quantity of lateral branches in the crop. Melatonin as a multi-regulatory phytohormone play vital functions in governing plant growth and development. It is synthesized from tryptophan via four key enzymes. Tryptophan decarboxylase (TDC), Tryptamine 5-hydroxylase (T5H), Serotonin N-acetyltransferase (SNAT), and N-Acetylserotonin O-methyltransferase (ASMT). Although the significance of melatonin is recognized, its impact on the growth and development of carnation remains understudied. In the current study, we investigated the effect of exogenous melatonin at different concentrations, on growth pattern of carnation, followed by genome-wide characterization, In-silico analysis and expression profiling of melatonin biosynthetic pathway genes. Results showed increased branching and reduced height with increased melatonin concentrations up to a point. In-silico analysis identified ten genes in the melatonin biosynthetic pathway, including two TDC, two T5H, one SNAT, and five ASMT members. Domain analysis confirmed the presence of characteristic domains such as pyridoxal-dependent decarboxylase, cytochrome P450, Acetyltransf_1, and O-methyltransferase. Physiochemical properties, gene structure, conserved motifs, promoter regions, gene ontology, synteny, and evolutionary relationships through phylogeny were also analysed. Sub-cellular localization predictions showed uniform distribution of melatonin biosynthetic enzymes across various cellular compartments. Expression analysis of these genes under different exogenous melatonin concentrations (100, 200, 300, 400, 500, and 1000 µM) revealed significant upregulation at 100 µM and 500 µM, while no change was observed at 1000 µM. These findings suggest that optimal exogenous melatonin concentrations enhance the expression of biosynthetic pathway genes ultimately led to increased branching in carnation due to increased endogenous melatonin levels. This study establishes a basis for future functional characterization of melatonin biosynthetic pathway genes to elucidate their roles in carnation growth and development.

Phosphorylation of cytochrome c at tyrosine 48 finely regulates its binding to the histone chaperone SET/TAF-Iβ in the nucleus.

Post-translational modifications (PTMs) of proteins are ubiquitous processes present in all life kingdoms, involved in the regulation of protein stability, subcellular location and activity. In this context, cytochrome c (Cc) is an excellent case study to analyze the structural and functional changes induced by PTMS as Cc is a small, moonlighting protein playing different roles in different cell compartments at different cell-cycle stages. Cc is actually a key component of the mitochondrial electron transport chain (ETC) under homeostatic conditions but is translocated to the cytoplasm and even the nucleus under apoptotic conditions and/or DNA damage. Phosphorylation does specifically alter the Cc redox activity in the mitochondria and the Cc non-redox interaction with apoptosis-related targets in the cytoplasm. However, little is known on how phosphorylation alters the interaction of Cc with histone chaperones in the nucleus. Here, we report the effect of Cc Tyr48 phosphorylation by examining the protein interaction with SET/TAF-Iβ in the nuclear compartment using a combination of molecular dynamics simulations, biophysical and structural approaches such as isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) and in cell proximity ligation assays. From these experiments, we infer that Tyr48 phosphorylation allows a fine-tuning of the Cc-mediated inhibition of SET/TAF-Iβ histone chaperone activity in vitro. Our findings likewise reveal that phosphorylation impacts the nuclear, stress-responsive functions of Cc, and provide an experimental framework to explore novel aspects of Cc post-translational regulation in the nucleus.

Abstract C038: Harnessing NK cells to improve targeted therapy therapeutic efficacy in BRAF- mutant melanoma

Abstract Malignant melanoma is the most lethal form of skin cancer, with BRAFV600E as the most common driver mutation. Despite the success of BRAF/MEK inhibitors (BRAF/MEKi) as a standard-of-care, drug resistance occurs at high frequency and remains a concern to be overcome. In addition to suppressing cancer cell proliferation and survival, BRAF/MEKi treatment alters the tumor microenvironment to favor anti-tumor immunity, with extensive previous studies focusing on the T cell compartment. Investigating other immune components, such as natural killer (NK) cells, may offer new insights into improving the overall effectiveness of BRAF/MEKi. NK cells are innate immune cells that provide activating signals to cytotoxic T cells, in addition to their critical roles in tumor surveillance and metastasis. However, the role of NK cells as a component of the BRAF/MEKi response in melanoma remains largely unknown. To investigate the role of NK cells in BRAF/MEKi-mediated tumor control, we depleted NK cells in melanoma-bearing mice. We found that the tumors developed full resistance faster in the NK cell-depleted group than in controls. Moreover, quantification of tumor-infiltrating NK cells in different melanoma phases, including off-therapy growth, therapy-induced regression, drug- tolerant residual disease, and on-therapy regrowth and resistance, revealed a positive correlation between NK cell infiltration and the tumor response to BRAF/MEKi. We further increased NK cell infiltration during drug-tolerant residual disease by inhibiting Ptpn22, a potential negative regulator of activity in NK and other immune cell populations, resulting in a delayed onset of therapy resistance. These results demonstrate the indispensable role of NK cells in the BRAF/MEKi tumor response and their potential to improve the therapeutic efficacy of BRAF/MEKi. Citation Format: Chia-Hsin Hsu, Andrew C. White. Harnessing NK cells to improve targeted therapy therapeutic efficacy in BRAF- mutant melanoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C038.

Abstract C047: AGX101: Preclinical evaluation of a TM4SF1-directed tubulin inhibitor conjugate with ongoing first-in-human trial

Abstract Introduction: TM4SF1 (Transmembrane-4 L-Six-Family-Member-1) is an endothelial marker with critical roles in angiogenesis, as well as a tumor cell antigen that contributes significantly to invasion and metastasis. TM4SF1 is upregulated 20-fold in angiogenic tumor vascular endothelium compared to normal vasculature endothelium and exhibits a unique nuclear internalization pathway. AGX101 is a novel tubulin inhibitor conjugate specifically directed against TM4SF1, delivering a potent maytansinoid payload directly to the nucleus of cells within the tumor microenvironment. Delivery to both the vascular and tumor cell compartments of the tumor results in three mechanisms of action (MoAs): (1) activation of tumor immune surveillance, (2) tumor blood supply deprivation, and (3) direct tumor cell killing. Methods: TM4SF1 expression was assessed across solid tumor samples using immunohistochemistry. Safety and pharmacokinetics of AGX101 were evaluated in non-human primates, with escalating doses to determine the highest non-severely toxic dose (HNSTD). Efficacy studies were also conducted in mouse models, enabling assessment of the minimum effective dose (MED) needed to engage each of the three MoAs. The combination of HNSTD and MED enables calculation of a therapeutic index (TI). Preclinical efficacy studies used either AGX101 (in human tumor xenograft models, to study the tumor cell killing MoA) or AGXB01, a rodent surrogate of AGX101, to study vascular and immune MoAs and all three MoAs in syngeneic mouse cancer models. The potential for synergy with immune checkpoint inhibitors (ICIs) was also investigated. A first-in-human study of AGX101 in patients with advanced solid tumors with the primary objective of safety and dose-limiting toxicities (DLTs) has initiated. Results: TMFS41 is broadly expressed across solid tumors with increasing scoring intensity in higher grade tumors. In non-human primates, AGX101 exhibited a favorable safety profile, being tolerated at relatively high doses and with dose dependent, monitorable, and reversible effects. In preclinical efficacy studies, AGXB01 and AGX101 as monotherapies demonstrated robust efficacy through each of the three MoAs in multiple syngeneic, xenograft, and orthotopic tumor models in mice and rats as well as non-tumor models of angiogenesis. Measured by exposure, TI was large. Additionally, AGXB01 was shown to potentiate the effects of ICIs in syngeneic mouse models (CT26, Renca, and B16-F10), suggesting a potential synergistic approach in cancer therapy. The first dose level of AGX101 monotherapy in humans has cleared without DLTs. Conclusion: AGX101, targeting the TM4SF1 antigen, represents a promising new approach in cancer therapy. The preclinical data suggest that AGX101 could provide a significant therapeutic benefit by novel and differentiated mechanisms of action, namely selectively targeting the tumor vasculature and potentiating immune-based therapies. Further clinical development of AGX101 is ongoing. Citation Format: Ildefonso Ismael Rodriguez Rivera, Meredith S. Pelster, Shou-Ching Jaminet, Paul Jaminet, Glen J. Weiss. AGX101: Preclinical evaluation of a TM4SF1-directed tubulin inhibitor conjugate with ongoing first-in-human trial [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C047.

Integrated proteomics and metabolomics analyses reveal new insights into the antitumor effects of valproic acid plus simvastatin combination in a prostate cancer xenograft model associated with downmodulation of YAP/TAZ signaling.

Despite advancements in therapeutic approaches, including taxane-based chemotherapy and androgen receptor-targeting agents, metastatic castration-resistant prostate cancer (mCRPC) remains an incurable tumor, highlighting the need for novel strategies that can target the complexities of this disease and bypass the development of drug resistance mechanisms. We previously demonstrated the synergistic antitumor interaction of valproic acid (VPA), an antiepileptic agent with histone deacetylase inhibitory activity, with the lipid-lowering drug simvastatin (SIM). This combination sensitizes mCRPC cells to docetaxel treatment both in vitro and in vivo by targeting the cancer stem cell compartment via mevalonate pathway/YAP axis modulation. Here, using a combined proteomic and metabolomic/lipidomic approach, we characterized tumor samples derived from 22Rv1 mCRPC cell-xenografted mice treated with or without VPA/SIM and performed an in-depth bioinformatics analysis. We confirmed the specific impact of VPA/SIM on the Hippo-YAP signaling pathway, which is functionally related to the modulation of cancer-related extracellular matrix biology and metabolic reprogramming, providing further insights into the molecular mechanism of the antitumor effects of VPA/SIM. In this study, we present an in-depth exploration of the potential to repurpose two generic, safe drugs for mCRPC treatment, valproic acid (VPA) and simvastatin (SIM), which already show antitumor efficacy in combination, primarily affecting the cancer stem cell compartment via MVP/YAP axis modulation. Bioinformatics analysis of the LC‒MS/MS and 1H‒NMR metabolomics/lipidomics results confirmed the specific impact of VPA/SIM on Hippo-YAP.

Subcellular mRNA kinetic modeling reveals nuclear retention as rate-limiting.

Eukaryotic mRNAs are transcribed, processed, translated, and degraded in different subcellular compartments. Here, we measured mRNA flow rates between subcellular compartments in mouse embryonic stem cells. By combining metabolic RNA labeling, biochemical fractionation, mRNA sequencing, and mathematical modeling, we determined the half-lives of nuclear pre-, nuclear mature, cytosolic, and membrane-associated mRNAs from over 9000 genes. In addition, we estimated transcript elongation rates. Many matured mRNAs have long nuclear half-lives, indicating nuclear retention as the rate-limiting step in the flow of mRNAs. In contrast, mRNA transcripts coding for transcription factors show fast kinetic rates, and in particular short nuclear half-lives. Differentially localized mRNAs have distinct rate constant combinations, implying modular regulation. Membrane stability is high for membrane-localized mRNA and cytosolic stability is high for cytosol-localized mRNA. mRNAs encoding target signals for membranes have low cytosolic and high membrane half-lives with minor differences between signals. Transcripts of nuclear-encoded mitochondrial proteins have long nuclear retention and cytoplasmic kinetics that do not reflect co-translational targeting. Our data and analyses provide a useful resource to study spatiotemporal gene expression regulation.

Bioactivity of Grape Pomace Extract and Sodium Selenite, Key Components of the OenoGrape Advanced Complex, on Target Human Cells: Intracellular ROS Scavenging and Nrf2/ARE Induction Following In Vitro Intestinal Absorption

Oenobiol Sun Expert, a food formulation designed to enhance skin health prior to sun exposure, has been optimized by incorporating the OenoGrape Advanced Complex, which includes grape pomace extract, increased selenium content and 10% lycopene-rich tomato extract, with these constituents exhibiting high antioxidant potential. To evaluate the effects of these individual ingredients and the overall formulation at the cellular level, the AOP1 cell antioxidant efficacy assay was employed to measure the intracellular free radical scavenging activity, while the Cell Antioxidant Assay (CAA or DCFH-DA) assay was used to assess peroxidation scavenging at the plasma membrane level. The indirect antioxidant activity was examined using stably transfected cell lines containing a luciferase reporter gene controlled by the Antioxidant Response Element (ARE), which activates the endogenous antioxidant system via the Nrf2/Keap1-ARE pathway. Our results indicate that among the individual components, grape pomace extract and sodium selenite possess high and complementary antioxidant properties. Grape pomace extract was particularly effective in inhibiting free radicals (AOP1 EC50 = 6.80 μg/mL) and activating the ARE pathway (ARE EC50 = 231.1 μg/mL), whereas sodium selenite exerted its effects through potent ARE activation at sub-microgram levels (EC50 = 0.367 μg/mL). In contrast, the lycopene-rich tomato extract did not show a notable contribution to the antioxidant effects. The antiradical activity of the OenoGrape Advanced Complex, comprising these three ingredients, was very efficient and consistent with the results obtained for the individual components (AOP1 EC50 = 15.78 µg/mL and ARE EC50 of 707.7 μg/mL). Similarly, the free radical scavenging activity still persisted in the Oenobiol Sun Expert formulation (AOP1 EC50 = 36.63 µg/mL). Next, in vitro intestinal transepithelial transfer experiments were performed. The basolateral compartments of cells exposed to the ingredients were collected and assessed using the same antioxidant cell assays. The direct and indirect antioxidant activities were measured on both hepatocytes and keratinocytes, demonstrating the bioavailability and bioactivity of grape pomace extract and sodium selenite. These finding suggest that the ingredients of this food supplement contribute to enhanced cytoprotection following ingestion.

Early-life thymectomy leads to an increase of granzyme-producing γδ T cells in children with congenital heart disease.

Congenital heart disease (CHD) is the most common birth defect in newborns, often requiring cardiac surgery with concomitant thymectomy that is known to increase disease susceptibility later in life. Studies of γδ T cells, which are one of the dominant T cells in the early fetal human thymus, are rare. Here, we provide a comprehensive analysis of the γδ T cell compartment via flow cytometry and next-generation sequencing in children and infants with CHD, who underwent cardiac surgery shortly after birth. A perturbation of the γδ T cell repertoire is evident, and Vδ1 T cell numbers are reduced. However, those cells that are present, do retain cytotoxicity. In contrast, GZMA+CD28+CD161hi innate effector Vγ9Vδ2 T cells are found in higher proportions. TCR-seq identifies an increase in TRDJ3+ γδ T cell clones in children with CHD, but not in a confirmatory group of neonates prior to CHD surgery, which overall points to a persistence of fetal-derived effector γδ T cells in children with CHD.

Phase angle and body composition in long-term type 1 diabetes in adults: a comparative study in a Brazilian public reference outpatient clinic.

Type 1 Diabetes Mellitus (DM1) affects a small percentage of the population. Nevertheless, its prevalence is currently growing with alarming data on uncontrolled cases. The importance of body composition and Phase Angle (PA), assessed by Bioelectrical Impedance (BIA), in long- term DM1 patients lies in the fact that alterations in cellular integrity and body compartments may affect risk profiles and metabolic control. The objective of this study was to compare PA and body composition parameters between adults with DM1 and healthy controls. A comparative study was carried out in a public university outpatient clinic including a cohort of adult patients of both sexes diagnosed with DM1 and healthy controls matched by age and sex in a 2:1 ratio. Anthropometric measurements included weight, height and BMI. Using the raw BIA data of Resistance and Reactance, fat-free mass (FFM), fat mass (FM), fat-free mass index (FFMI), fat mass index (FMI), PA and standardized PA (SPA) were calculated. Means or medians were compared between the groups. Regression models were used to identify distinguishing characteristics of the groups and associations within the DM1 group (i.e. glycated hemoglobin (HbA1c), disease duration, presence of microvascular complications, capillary blood glucose, BMI and FMI). 88 patients with DM1and 46 healthy controls were evaluated. PA (6.05 vs. 6.85, p = 0.000) and SPA (-1.47 vs. -0,37, p = 0.000) were lower in patients with DM1 compared to healthy controls. People with DM1 displayed higher adiposity (%FM = 29.6 vs. 27.6, p = 0.016; FMI = 7.00 vs. 6.33, p = 0.016) and lower %FFM compared to healthy controls. Most of the differences were maintained after sex stratification; however, men with DM1 showed a lower FFMI than male controls (18.2 vs. 20.16, p = 0.029). Patients with DM1 present lower PA than healthy controls, which may be related to worse cell membrane integrity. Significant body composition differences between the groups and between sexes were identified, with data showing greater adiposity in women with DM1 and men displaying lower muscle mass. These findings suggest the importance of including PA and body composition evaluations in the follow-up of patients with DM1. The ultimate goal is to obtain a better metabolic control and, consequently, a better prognosis.

Sedimentology of False Bay (Western Cape, South Africa): geological background information for the integrated environmental management of a physically confined coastal compartment

Abstract False Bay is a large, physically confined embayment located along the southwest coast of South Africa. It is a classic example of a coastal compartment or littoral cell that acts as a receptor and sink of both terrestrial (siliciclastic) and marine (bioclastic) material. It is almost square in shape, measuring ∼35 km N-S and ∼39 km W-E and covering ∼1130 km2. Only a few small rivers discharge into the bay, as a consequence of which only small amounts of sediment are episodically supplied to the bay. The bathymetry reveals two well-defined terraces, one between 30 and 45 m, the other between 50 and 55 m water depth, which are indicative of extended Pleistocene sea-level stillstands. Sediment is dispersed by long-period ocean swells approaching from the southwestern quadrant, as well as nearshore swell- and wind-driven currents. The presence of a prominent rock pinnacle at the entrance to the bay (Rocky Bank) causes wave orthogonals to converge on its leeward side, and which results in substantial wave amplification along the eastern shore of the bay. Coarse-grained sediments (gravel, very coarse sand, coarse sand and medium sand) line the rocky shores in the west and east, and are dispersed around and away from submarine rock outcrops, whereas fine-grained sediments (fine sand, very fine sand and mud) are largely confined to an arcuate belt extending along the centre of the bay from shallow water in the northwest to the deepest parts in the south. This is documented by the distribution of individual size fractions and the mean grain size, and applies to both the siliciclastic and the bioclastic sediment components which appear to be in hydraulic equilibrium. There is a distinct N-S gradient from lower to higher bioclastic content with increasing water depth. The textural parameters (mean grain size, sorting and skewness) reveal the existence of two hydraulic populations that are mixed in various proportions in the course of their dispersal in the form of either bed load or suspended load transport. The mud fraction is closely linked to the dispersal pattern of the fine and very fine sand fractions, indicating that it is transported in the form of aggregates and/or faecal pellets, the bulk of which is evidently in hydraulic equilibrium with the finer-grained sands. It can be anticipated that, during lower Pleistocene sea levels, the palaeo-False Bay valley was occupied, or at least regularly visited, by large and small African mammals and early humans.

Abstract A008: Adaptive immune receptor repertoire characterization highlights the importance of interplay between B and T cells in disease progression

Abstract Understanding the adaptive immune system is integral to disease progression studies, development of diagnostic biomarkers, identification of therapeutic targets, and discerning varied treatment responses. Despite growing interest in profiling B and T cells in cancer research, the interaction between these cells and also the crucial roles of γδ T cells often go overlooked. In this research project, we explored the role of crucial yet frequently ignored γδ T cells in cancer research, using immune repertoire data from bladder cancer patients. We also studied the synergy between B and T cells in the immune repertoires of COVID-19 patients.Roche has developed immunoPETE (immune repertoire Primer Extension Target Enrichment), a DNA- based target enrichment assay to capture adaptive immune repertoire data*. immunoPETE efficiently identifies the heavy chains of B cells, alongside αβ and γδ T cells in one reaction, facilitating normalized clone counts across the adaptive immune system. To explore the significance of γδ T cells, we profiled the immune repertoires of 24 Non-Muscle Invasive Bladder Cancer patients treated with Bacillus Calmette-Guerin (BCG). Additionally, we evaluated the interplay between B and T cells in 117 peripheral blood immune repertoire from healthy and COVID-19 afflicted individuals, ranging from mild to severe cases.Immune repertoire data analysis indicates that underrepresentation of T cell receptor delta locus (TRD) is associated with higher risk of bladder cancer recurrence or progress. Urine pellet DNA was available for a subset of the patients in this study. Comparing immune repertoires from PBMC and urine revealed that urine repertoires represent the tumor repertoire more accurately than PBMC. Analysis of the immune repertoires from the COVID-19 patients linked disease severity with immune repertoire measures across all three cell types, highlighting the importance of multiple lymphocyte classes in disease progression. Using immunoPETE technology, which is capable of capturing the heavy chain of multiple immune cell types simultaneously in a single reaction, we have demonstrated that both B cell and T cell compartments provide distinct and valuable information in cancer and infectious diseases, each responding uniquely to the disease. Additionally, we emphasized the significance of γδ T cells in predicting cancer prognosis. Note: immunoPETE is for Research Use Only. Not for use in diagnostic procedures. Citation Format: Hossein Asgharian, Hamid Mirebrahim, Dilduz Telman, Hahn Zhao, Ulrich Schlecht, Sylvie McNamara, Rajiv Dua, Jan Berka, Patrick Bogard, Dinesh Kumar. Adaptive immune receptor repertoire characterization highlights the importance of interplay between B and T cells in disease progression [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A008.

Intracellular Mg2+ concentrations are differentially regulated in the sperm head and mid-piece in acrosome reaction inducing conditions.

The sperm ability to fertilize involves the regulation of ATP levels. Because inside cells, ATP is complexed with Mg2+ ions, changes in ATP levels result in changes in intracellular Mg2+ concentration ([Mg2+]i), which can be followed using intracellular Mg2+ sensors such as Mag-520. In this work, we tested conditions known to decrease sperm ATP such as starvation and capacitation. As expected, in these conditions [Mg2+]i increased in all cell compartments. In contrast, when ATP increases, such as adding nutrients to starved sperm, [Mg2+]i significantly decreases in all compartments. On the other hand, when the acrosome reaction was induced, either with progesterone or with ionomycin, [Mg2+]i was differentially regulated in the head and mid-piece. While Mag-520 fluorescence increased in the sperm mid-piece, it decreased in the head. These changes were observed in capacitated as well as in starved sperm but not in sperm incubated in conditions that do not support capacitation. Changes in [Mg2+]i were still observed when the sperm were incubated in high extracellular Mg2+ suggesting that this decrease is not due to Mg2+ efflux. Interestingly, the progesterone and ionomycin effects on [Mg2+]i were abolished on sperm incubated in Ca2+-free media. Altogether, these results indicate that [Mg2+]i is regulated in sperm during capacitation and acrosomal reaction, and suggest that these measurements can serve to evaluate ATP levels in real time.

In utero human cytomegalovirus infection expands NK-like FcγRIII+ CD8+ T cells that mediate Fc antibody functions.

Human cytomegalovirus (HCMV) profoundly impacts host T and natural killer (NK) cells across the lifespan, yet how this common congenital infection modulates developing fetal immune cell compartments remains underexplored. Using cord blood from neonates with and without congenital HCMV (cCMV) infection, we identify an expansion of Fcγ receptor III (FcγRIII)-expressing CD8+ T cells following HCMV exposure in utero. Most FcγRIII+ CD8+ T cells express the canonical αβ T cell receptor (TCR) but a proportion express non-canonical γδ TCR. FcγRIII+ CD8+ T cells are highly differentiated and have increased expression of NK cell markers and cytolytic molecules. Transcriptional analysis reveals FcγRIII+ CD8+ T cells upregulate T-bet and downregulate BCL11B, known transcription factors that govern T/NK cell fate. We show that FcγRIII+ CD8+ T cells mediate antibody-dependent IFNγ production and degranulation against IgG-opsonized target cells, similar to NK cell antibody-dependent cellular cytotoxicity (ADCC). FcγRIII+ CD8+ T cell Fc effector functions were further enhanced by interleukin-15 (IL-15), as has been observed in neonatal NK cells. Our study reveals that FcγRIII+ CD8+ T cells elicited in utero by HCMV infection can execute Fc-mediated effector functions bridging cellular and humoral immunity and may be a promising target for antibody-based therapeutics and vaccination in early life.

TRBC1-CAR T cell therapy in peripheral T cell lymphoma: a phase 1/2 trial.

Relapsed/refractory peripheral T cell lymphomas (PTCLs) are aggressive tumors with a poor prognosis. Unlike B cell lymphomas, treatment of PTCL has not benefited from advances in immunotherapy. This is largely due to a lack of suitable target antigens that discriminate malignant from normal T cells, thus avoiding severe immunosuppression consequent to depletion of the entire T cell compartment. We recently described a targeting strategy based on the mutually exclusive expression of T cell antigen receptor beta-chain constant domain (TRBC) 1 and 2. Selective targeting of the T cell antigen receptor beta-chain expressed by the (clonal) malignancy spares normal T cells expressing the other chain. The LibraT1 study is an ongoing, multicenter, international, single-arm phase 1/2 study of TRBC1-directed autologous chimeric antigen receptor (CAR) T cells (AUTO4) in relapsed/refractory TRBC1-positive PTCL. Primary objectives were assessment of safety and tolerability of AUTO4 infusion. Key secondary endpoints included efficacy, CAR T cell expansion and persistence. Here we describe the findings from dose escalation in LibraT1 in the first ten patients, in a non-prespecified interim analysis. AUTO4 resulted in low frequency of severe immunotoxicity, with one of ten patients developing grade 3 cytokine release syndrome. Complete metabolic response was observed in four of ten evaluable patients, with remissions being durable beyond 1 year in two patients. While an absence of circulating CAR T cells was observed, CAR T cells were readily detected in lymph node biopsy samples from sites of original disease suggesting homing to tumor sites. These results support the continuing exploration of TRBC1 targeting in PTCL. ClinicalTrials.gov registration: NCT03590574 .

TMIC-65. USING BIOINFORMATICS TO INVESTIGATE THE TUMOR MICROENVIRONMENT OF DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas, H3K27-altered (DMG) are highly malignant brain tumors with a 2-year survival rate of less than 10 percent. These tumors are primarily localized to the pons and thalamus regions of the brain. The cellular compartment of the tumor microenvironment (TME) which surrounds a tumor is composed of tumor and non-tumor cells, such as immune cells and oligodendrocytes. Communication between tumor cells and non-tumor cells in the TME can promote tumor growth and targeting communication networks could improve patient outcomes. Using a bioinformatics approach and publicly available pediatric DMG single-cell RNA-sequence (scRNA-seq) data, several methods of communication (secreted signaling, cell-cell contact, and extracellular matrix) within the DMG TME were identified. Based on the high communication probability, outgoing and incoming networks from non-tumor cells and tumor cells were inferred, such as the Complement, Galectin, and CD96 pathways. Specific ligand-receptor interactions between non-tumor and tumor cells were also found, including LGALS9-HAVCR2, PDGFB/PDGFRA, and COL4A5/ITGAV. A subsequent literature review was performed to validate these identified pathways in other tumors, including gliomas, and inform future in vitro experiments with primary DMG cell lines.

TMIC-26. IMMUNE CELL DYNAMICS WITHIN GLIOMAS: THE INFLUENCE OF GRADE AND IDH STATUS

Abstract Mutations in isocitrate dehydrogenase (IDH) are known to significantly impact the prognosis and biology of diffuse gliomas. A number of studies have compared the immune microenvironment of IDH-wildtype glioma versus IDH-mutant glioma, and have also characterized the impact of mutant-IDH on lymphocytes. However, few studies have explored how mutant-IDH may affect tumors as they progress and increase in grade, or have delved into the myeloid immune cell compartment. Thus, we sought to understand whether tumor progression affected intratumoral immune cell composition within IDH-mutant glioma, and aimed to compare IDH-mutant versus IDH-wildtype glioma in both humans and mice. Using bulk RNA sequencing, we compared paired samples from patients with IDH-mutant glioma before and after progression, focusing on differentially-expressed immunological genes and relative immune cell proportions. We also compared grade 4 IDH-mutant astrocytoma cases to IDH-wildtype glioblastoma, performing both transcriptomic and cellular analyses. Finally, we engineered murine glioma cells to test the effect of mutant-IDH alone in the tumor microenvironment. We found that multiple immune-related genes distinguish low- and high-grade IDH-mutant glioma, and that high-grade tumors contain greater proportions of suppressive myeloid cells. When comparing IDH-wildtype to IDH-mutant glioma, we found enrichment of suppressive macrophages and myeloid-derived suppressor cells (MDSC) in IDH-wildtype tumors. Finally, our murine model of IDH-wildtype versus IDH-mutant glioma mirrored many of the myeloid cell shifts observed in human tumors. Our data show that progression of IDH-mutant tumors is a significant immunological phenomenon, associated with marked alterations in the tumor immune microenvironment. We also show that IDH-mutant glioma harbors greater inflammatory signatures and fewer suppressive myeloid cells than IDH-wildtype glioma, and that engineering mutant IDH into murine tumors is sufficient to model these differences. These findings improve our understanding of key intratumoral cell populations that may be harnessed by future immunotherapeutic strategies.

Dynamic reorganization of multivesicular bodies and exosome production impacted by sonoporation

Naturally occurring cell-derived extracellular vesicles (EVs) have emerged as attractive nanocarriers for drug delivery. However, production of large quantities of EVs for clinical applications in a scalable manner remains a significant challenge. This study investigated at the single cell level how sonoporation, or membrane poration produced by ultrasound-induced microbubble cavitation, impacts EV production using mouse macrophage RAW 264.7 cells stably expressing CD63-GFP as a model system. Real-time fluorescence videomicroscopy detected rapid changes in CD63-GFP, a tetraspanin family member highly enriched in intraluminal vesicles tagged with GFP, to track changes in multivesicular bodies (MVBs), which are the cellular compartments where exosomes originate within the cells. Our results revealed distinct dynamic changes in CD63-GFP intensity and distribution in RAW 264.7 cells in terms of response time and duration depending on whether the cells were directly or indirectly impacted by sonoporation, suggesting reorganization of MVBs in response to direct and indirect mechanisms resulted from the mechanical impact of ultrasound pulse on the cells. Analysis of the supernatant from sonoporation-treated RAW 264.7 cells expressing CD63-GFP demonstrated a delayed and sustained increase in the production of CD63-GFP-positive EVs. These results show the robust and detailed effect of sonoporation and reveal insights into sonoporation-induced EV release useful for guiding the application of sonoporation to enhance large-scale EV production.

Forced Overexpression and Knockout Analysis of SLC30A and SLC39A Family Genes Suggests Their Involvement in Establishing Resistance to Cisplatin in Human Cancer Cells

Abstract: The metabolism of zinc and manganese plays a pivotal role in cancer progression by mediating cancer cell growth and metastasis. The SLC30A family proteins SLC30A3 and SLC30A10 mediate the efflux of zinc, manganese, and probably other transition element ions outside the cytoplasm to the extracellular space or into intracellular membrane compartments. The SLC39A family members SLC39A8 and SLC39A14 are their functional antagonists that transfer these ions into the cytoplasm. Recently, the SLC30A10 gene was suggested as a promising methylation biomarker of colorectal cancer. Here, we investigated whether forced overexpression or inactivation of SLC30A and SLC39A family genes has an impact on the phenotype of cancer cells and their sensitivity to cancer therapeutics. In the human colon adenocarcinoma HCT-15 and duodenal adenocarcinoma HuTu80 cell lines, we generated clones with knockouts of the SLC39A8 and SLC39A14 genes and forced overexpression of the SLC30A3, SLC30A10, and SLC39A8 genes. Gene expression in the mutant and control cells was assessed by RNA sequencing. The cell growth rate, mitochondrial activity, zinc accumulation, and sensitivity to the drugs cetuximab and cisplatin were investigated in functional tests. Overexpression or depletion of SLC30A or SLC39A family genes resulted in the deep reshaping of intracellular signaling and provoked hyperactivation of mitochondrial respiration. Variation in the expression of the SLC30A/SLC39A genes did not increase the sensitivity to cetuximab but significantly altered the sensitivity to cisplatin: overexpression of SLC30A10 resulted in an ~2.7–4 times increased IC50 of cisplatin, and overexpression of SLC30A3 resulted in an ~3.3 times decreased IC50 of cisplatin. The SLC30A/SLC39A genes should be considered as potential cancer drug resistance biomarkers and putative therapeutic targets.

Antagonistic Effects of Actin-Specific Toxins on Salmonella Typhimurium Invasion into Mammalian Cells

Competition between bacterial species is a major factor shaping microbial communities. It is possible but remains largely unexplored that competition between bacterial pathogens can be mediated through antagonistic effects of bacterial effector proteins on host systems, particularly the actin cytoskeleton. Using Salmonella Typhimurium invasion into cells as a model, we demonstrate that invasion is inhibited if the host actin cytoskeleton is disturbed by actin-specific toxins, namely, Vibrio cholerae MARTX actin crosslinking (ACD) and Rho GTPase inactivation (RID) domains, Photorhabdus luminescens TccC3, and Salmonella’s own SpvB. We noticed that ACD, being an effective inhibitor of tandem G-actin-binding assembly factors, is likely to inhibit the activity of another Vibrio effector, VopF. In reconstituted actin polymerization assays and by live-cell microscopy, we confirmed that ACD potently halted the actin nucleation and pointed-end elongation activities of VopF, revealing competition between these two V. cholerae effectors. These results suggest that bacterial effectors from different species that target the same host machinery or proteins may represent an effective but largely overlooked mechanism of indirect bacterial competition in host-associated microbial communities. Whether the proposed inhibition mechanism involves the actin cytoskeleton or other host cell compartments, such inhibition deserves investigation and may contribute to a documented scarcity of human enteric co-infections by different pathogenic bacteria.

Visualization of endogenous G proteins on endosomes and other organelles

Classical G-protein-coupled receptor (GPCR) signaling takes place in response to extracellular stimuli and involves receptors and heterotrimeric G proteins located at the plasma membrane. It has recently been established that GPCR signaling can also take place from intracellular membrane compartments, including endosomes that contain internalized receptors and ligands. While the mechanisms of GPCR endocytosis are well understood, it is not clear how well internalized receptors are supplied with G proteins. To address this gap, we use gene editing, confocal microscopy, and bioluminescence resonance energy transfer to study the distribution and trafficking of endogenous G proteins. We show here that constitutive endocytosis is sufficient to supply newly internalized endocytic vesicles with 20–30% of the G protein density found at the plasma membrane. We find that G proteins are present on early, late, and recycling endosomes, are abundant on lysosomes, but are virtually undetectable on the endoplasmic reticulum, mitochondria, and the medial-trans Golgi apparatus. Receptor activation does not change heterotrimer abundance on endosomes. Our findings provide a subcellular map of endogenous G protein distribution, suggest that G proteins may be partially excluded from nascent endocytic vesicles, and are likely to have implications for GPCR signaling from endosomes and other intracellular compartments.