Natural Killer (NK) cells are cytotoxic innate lymphocytes able to detect transformed cells through the balanced action of inhibitory and activating receptors. NKG2D is one of the main activating receptors involved in tumor surveillance thanks to its ability to recognize stress-induced ligands. Of note, the prolonged exposure to NKG2D ligands promotes receptor down-modulation that results in defective activation of NKG2D and other unrelated activating receptors, including DNAM-1 that is also involved in tumor clearance. However, further investigations are necessary to characterize how the NKG2D/DNAM-1 interplay affects NK cell anti-tumor function.
Primary cultured human NK cells were stimulated with the natural ligand MICA or an anti-NKG2D agonist antibody. The expression of activating and inhibitory receptors as well as DNAM-1-triggered signaling events and cytotoxicity were evaluated by flow cytometry. DNAM-1-mediated granule polarization was evaluated by confocal microscopy.
We showed that NKG2D crosslinking mediated by the natural ligand MICA or an agonist antibody had different consequences on primary cultured human NK cells. Indeed, MICA stimulation increases the expression of the checkpoint receptor TIGIT that is able to counteract DNAM-1 activation. Stimulation with the agonist antibody, without altering TIGIT expression, directly inhibits DNAM-1-mediated signal transduction and cytotoxic function with a mechanism that required NKG2D endocytosis.
Our findings contribute to shed light on the functional consequences of NKG2D engagement, demonstrating that a direct impact on DNAM-1-mediated signal transduction occurs independently from the modality of NKG2D crosslinking. Understanding the molecular mechanisms responsible for suppression of NK cell activation may help the development of therapeutic anti-cancer strategies aimed to prevent NK cell dysfunction or to reinvigorate an impaired cytotoxic activity.
Copyright © 2025 Marangio, Milito, Putro, Carnevale, Capuano, Zingoni, Cippitelli, Santoni, Paolini and Molfetta.

BACKGROUNDPredicting immune effector cell-associated neurotoxicity syndrome (ICANS) in patients infused with CAR T cells is still a conundrum. This complication, thought to be consequent to CAR T cell activation, arises a few days after infusion, when circulating CAR T cells are scarce and specific CAR T cell-derived biomarkers are lacking.METHODSCAR+ extracellular vesicle (CAR+EV) release was assessed in human CD19.CAR T cells cocultured with CD19+ target cells. A prospective cohort of 100 patients with B cell lymphoma infused with approved CD19.CAR T cell products was assessed for plasma CAR+EVs as biomarkers of in vivo CD19.CAR T cell activation. Human induced pluripotent stem cell-derived (iPSC-derived) neural cells were used as a model for CAR+EV-induced neurotoxicity.RESULTSIn vitro release of CAR+EVs occurs within 1 hour after target engagement. Plasma CAR+EVs are detectable 1 hour after infusion. A concentration greater than 132.8 CAR+EVs/μL at hour +1 or greater than 224.5 CAR+EVs/μL at day +1 predicted ICANS in advance of 4 days, with a sensitivity and a specificity outperforming other ICANS predictors. ENO2+ nanoparticles were released by iPSC-derived neural cells upon CAR+EV exposure and were increased in plasma of patients with ICANS.CONCLUSIONPlasma CAR+EVs are an immediate signal of CD19.CAR T cell activation, are suitable predictors of neurotoxicity, and may be involved in ICANS pathogenesis.TRIAL REGISTRATIONNCT04892433, NCT05807789.FUNDINGLife Science Hub-Advanced Therapies (financed by Health Ministry as part of the National Plan for Complementary Investments to the National Recovery and Resilience Plan [NRRP]: E.3 Innovative health ecosystem for APC fees and immunomonitoring).

Background: Glioblastoma multiforme (GBM) is the most lethal malignancy in brain, which is surrounded by the blood-brain barrier (BBB), which limits the efficacy of standard treatments. Developing an effective drug that can penetrate the blood-brain barrier (BBB) remains a critical challenge in the fight against GBM. CC12 (NSC749232) is an anthraquinone tetraheterocyclic homolog with a lipophilic structure that may facilitate penetration of the brain area. Methods: We used temozolomide sensitive and resistance GBM cells and animal model to identify the CC12 delivery, anti-tumor potential and its underlying mechanism. Results: Importantly, toxicity triggered by CC12 was not associated with the methyl guanine-DNA methyl transferase (MGMT) methylation status which revealed a greater application potential compared to temozolomide. Alexa F488 cadaverine-labelled CC12 successfully infiltrated into the GBM sphere; in addition, 68Ga-labeled CC12 was also found in the orthotopic GBM area. After passing BBB, CC12 initiated both caspase-dependent intrinsic/extrinsic apoptosis pathways and apoptosis-inducing factor, EndoG-related caspase-independent apoptosis signaling in GBM. RNA sequence analysis from The Cancer Genome Atlas indicated that LYN was overexpressed in GBM is associated with poorer overall survival. We proved that targeting of LYN by CC12 may diminish GBM progression and suppress it downstream factors such as signal transduction and activator of extracellular signal-regulated kinases (ERK)/transcription 3 (STAT3)/nuclear factor (NF)-κB. CC12 was also found to participate in suppressing GBM metastasis and dysregulation of the epithelial-mesenchymal transition (EMT) through inactivation of the LYN axis. Conclusion: CC12, a newly developed BBB-penetrating drug, was found to possess an anti-GBM capacity via initiating an apoptotic mechanism and disrupting LYN/ERK/STAT3/NF-κB-regulated GBM progression.
© The author(s).

The genesis of SMAC mimetic drugs is founded on the observation that many cancers amplify IAP proteins to facilitate their survival, and therefore removal of these pathways would re-sensitize the cells towards apoptosis. It has become increasingly clear that SMAC mimetics also interface with the immune system in a modulatory manner. Suppression of IAP function by SMAC mimetics activates the non-canonical NF-κB pathway which can augment T cell function, opening the possibility of using SMAC mimetics to enhance immunotherapeutics.
We have investigated the SMAC mimetic LCL161, which promotes degradation of cIAP-1 and cIAP-2, as an agent for delivering transient costimulation to engineered BMCA-specific human TAC T cells. In doing so we also sought to understand the cellular and molecular effects of LCL161 on T cell biology.
LCL161 activated the non-canonical NF-κB pathway and enhanced antigen-driven TAC T cell proliferation and survival. Transcriptional profiling from TAC T cells treated with LCL161 revealed differential expression of costimulatory and apoptosis-related proteins, namely CD30 and FAIM3. We hypothesized that regulation of these genes by LCL161 may influence the drug's effects on T cells. We reversed the differential expression through genetic engineering and observed impaired costimulation by LCL161, particularly when CD30 was deleted. While LCL161 can provide a costimulatory signal to TAC T cells following exposure to isolated antigen, we did not observe a similar pattern when TAC T cells were stimulated with myeloma cells expressing the target antigen. We questioned whether FasL expression by myeloma cells may antagonize the costimulatory effects of LCL161. Fas-KO TAC T cells displayed superior expansion following antigen stimulation in the presence of LCL161, suggesting a role for Fas-related T cell death in limiting the magnitude of the T cell response to antigen in the presence of LCL161.
Our results demonstrate that LCL161 provides costimulation to TAC T cells exposed to antigen alone, however LCL161 did not enhance TAC T cell anti-tumor function when challenged with myeloma cells and may be limited due to sensitization of T cells towards Fas-mediated apoptosis.
Copyright © 2023 Afsahi, Silvestri, Moore, Graham, Bacchiochi, St-Jean, Baker, Korneluk, Beug, LaCasse and Bramson.

Licochalcone A (LicA), a major active component of licorice, has been reported to exhibit various pharmacological actions. The purpose of this study was to investigate the anticancer activity of LicA and detail its molecular mechanisms against ovarian cancer. SKOV3 human ovarian cancer cells were used in this study. Cell viability was measured using a cell counting kit-8 assay. The percentages of apoptotic cells and cell cycle arrest were determined by flow cytometry and Muse flow cytometry. The expression levels of proteins regulating cell apoptosis, cell cycle, and the signal transducer and activator of transcription 3 (STAT3) signaling pathways were examined using Western blotting analysis. The results indicated that LicA treatment inhibited the cell viability of SKOV3 cells and induced G2/M phase arrest. Furthermore, LicA induced an increase in ROS levels, a reduction in mitochondrial membrane potential, and apoptosis accompanied by an increase in cleaved caspases and cytoplasmic cytochrome c. Additionally, LicA caused a dramatic decrease in STAT3 protein levels, but not mRNA levels, in SKOV3 cells. Treatment with LicA also reduced phosphorylation of the mammalian target of rapamycin and eukaryotic translation initiation factor 4E-binding protein in SKOV3 cells. The anti-cancer effects of LicA on SKOV3 cells might be mediated by reduced STAT3 translation and activation.