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.

B7 homolog 3 (B7-H3), an overexpressed antigen across multiple solid cancers, represents a promising target for CAR T cell therapy. This study investigated the expression of B7-H3 across various solid tumors and developed novel monoclonal antibodies (mAbs) targeting B7-H3 for CAR T cell therapy.
Expression of B7-H3 across various solid tumors was evaluated using RNA-seq data from TCGA, TARGET, and GTEx datasets and by flow cytometry staining. B7-H3-specific mAbs were developed by immunizing mice with human B7-H3, screening with ELISA, and analyzing kinetics with surface plasmon resonance. These mAbs were used to create second-generation CAR constructs, which were evaluated in vitro and in vivo for their antitumor function.
We identified four mAb clones from immunized mice, with three demonstrating high specificity and affinity. The second-generation B7-H3 CAR T cells derived from these mAbs exhibited robust cytotoxicity against B7-H3-positive targets and successfully infiltrated and eliminated tumor spheroids in vitro. In a xenograft mouse model of glioblastoma, these CAR T cells, particularly those derived from clone A2H4, eradicated the primary tumor, and effectively controlled rechallenge tumor, resulting in prolonged survival of the xenograft mice. In vivo T cell trafficking revealed high accumulation and persistence of A2H4-derived CAR T cells at the tumor site.
Our results provide novel B7-H3-targeted CAR T cells with high efficacy, paving the way for clinical translation of solid tumor treatment.
© Author(s) (or their employer(s)) 2025. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group.

Triple-negative breast cancer (TNBC) shows an urgent need for new therapies. We discovered Ropporin-1 (ROPN1) as a target to treat TNBC with T-cells. ROPN1 showed high and homogenous expression in 90% of primary and metastatic TNBC but not in healthy tissues. HLA-A2-binding peptides were detected via immunopeptidomics and predictions and used to retrieve T-cell receptors (TCRs) from naïve repertoires. Following gene introduction into T-cells and stringent selection, we retrieved a highly specific TCR directed against the epitope FLYTYIAKV that did not recognize non-cognate epitopes from alternative source proteins. Notably, this TCR mediated killing of three-dimensional tumoroids in vitro and tumor cells in vivo and outperformed standard-of-care drugs. Finally, the T-cell product expressing this TCR and manufactured using a clinical protocol fulfilled standard safety and efficacy assays. Collectively, we have identified and preclinically validated ROPN1 as a target and anti-ROPN1 TCR T-cells as a treatment for the vast majority of TNBC patients.

PD-1 blockade has been approved for head and neck squamous cell carcinoma (HNSCC) patients. However, many HNSCC patients do not respond to this treatment, and other tumor microenvironmental factors may promote resistance to PD-1 blockade. We previously identified increased expression of the inhibitory receptor NKG2A on CD8+ T cells in HNSCC tumors compared with T cells in matching PBMC samples. Mechanisms that promote NKG2A expression and the role of NKG2A on human T cells in the tumor microenvironment, however, are uncertain. In this study, we show that tumor-conditioned media (TCM) of HNSCC cancer cell lines or ascites fluid from colorectal carcinoma patients is sufficient to induce the expression of NKG2A and other inhibitory receptors on activated CD8+ T cells isolated from PBMCs of healthy donors. Boiling or small molecular mass cutoff filtering did not eliminate the effect of TCM, suggesting that a small molecule promotes NKG2A. T cell activation in TCM decreased the basal and maximal mitochondrial respiration to metabolically restrain CD8+ T cells. Functionally, T cell activation in TCM reduced CD8+ T cell cytotoxicity as shown by lower production of cytokines, granzyme B, and perforin. Furthermore, TCM prevented CD8+ T cells from killing cancer cells in response to an anti-CD19/anti-CD3 bispecific T cell engager. Thus, a small secreted molecule from HNSCC cells can induce NKG2A expression and promote T cell dysfunction. Our findings may lead to targets for novel cancer therapies or biomarkers for NKG2A blockade response and provide a model to study T cell dysfunction and impaired metabolism.
Copyright © 2024 The Authors.

Tumor-infiltrating lymphocytes (TILs) targeting neoantigens can effectively treat a selected set of metastatic solid cancers. However, harnessing TILs for cancer treatments remains challenging because neoantigen-reactive T cells are often rare and exhausted, and ex vivo expansion can further reduce their frequencies. This complicates the identification of neoantigen-reactive T-cell receptors (TCRs) and the development of TIL products with high reactivity for patient treatment.
We tested whether TILs could be in vitro stimulated against neoantigens to achieve selective expansion of neoantigen-reactive TILs. Given their prevalence, mutant p53 or RAS were studied as models of human neoantigens. An in vitro stimulation method, termed "NeoExpand", was developed to provide neoantigen-specific stimulation to TILs. 25 consecutive patient TILs from tumors harboring p53 or RAS mutations were subjected to NeoExpand.
We show that neoantigenic stimulation achieved selective expansion of neoantigen-reactive TILs and broadened the neoantigen-reactive CD4+ and CD8+ TIL clonal repertoire. This allowed the effective isolation of novel neoantigen-reactive TCRs. Out of the 25 consecutive TIL samples, neoantigenic stimulation enabled the identification of 16 unique reactivities and 42 TCRs, while conventional TIL expansion identified 9 reactivities and 14 TCRs. Single-cell transcriptome analysis revealed that neoantigenic stimulation increased neoantigen-reactive TILs with stem-like memory phenotypes expressing IL-7R, CD62L, and KLF2. Furthermore, neoantigenic stimulation improved the in vivo antitumor efficacy of TILs relative to the conventional OKT3-induced rapid TIL expansion in p53-mutated or KRAS-mutated xenograft mouse models.
Taken together, neoantigenic stimulation of TILs selectively expands neoantigen-reactive TILs by frequencies and by their clonal repertoire. NeoExpand led to improved phenotypes and functions of neoantigen-reactive TILs. Our data warrant its clinical evaluation.
NCT00068003, NCT01174121, and NCT03412877.
© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.