Search Results (1,604)

Chimeric antigen receptor (CAR) T cell therapy has demonstrated clinical success in hematologic malignancies but has limited efficacy in solid tumors due to tumor microenvironment (TME) barriers that impede CAR T cell recognition, infiltration, and sustained function. Traditional 2D assays inadequately recapitulate these constraints, necessitating improved in vitro models. This study validated a 3D tumor spheroid platform using an agarose microwell system to generate uniform B7-H3-positive spheroids from multiple solid tumor cell lines, enabling the evaluation of CAR T cell activity. TME-relevant immune modulation under 3D conditions was analyzed by flow cytometry for B7-H3, MHC I/II, and antigen processing machinery (APM), followed by co-culture with B7-H3 CAR T cells to assess cytotoxicity, spheroid integrity, tumor viability, and CAR T cell activation, exhaustion, and cytokine production. Two human cancer-cell-line-derived spheroids, DU 145 (prostate cancer) and SUM159 (breast cancer), retained B7-H3 expression, while MC38 (mouse colon cancer)-derived spheroids served as a B7-H3 negative control. Under 3D culture conditions, DU 145 and SUM159 spheroids acquire TME-like immune evasion characteristics and specifically downregulated MHC-I and APM (TAP1, TAP2, LMP7) with concurrent upregulation of MHC-II and calreticulin. Co-culture showed effective spheroid infiltration, cytotoxicity, and structural disruption, with infiltrating CAR T cells displaying higher CD4⁺ fraction, activation, exhaustion, effector/terminal differentiation, and IFN-γ/TNF-α production. This 3D platform recapitulates critical TME constraints and provides a cost-effective, feasible preclinical tool to assess CAR T therapies beyond conventional 2D assays. Full article

Chimeric antigen receptor (CAR)-T cell therapy has emerged as a transformative form of immunotherapy, enabling the precise engineering of T cells to recognize and eliminate pathogenic cells. In hematologic malignancies, CAR-T cells targeting CD19 or B cell maturation antigens have achieved remarkable remission rates and durable responses in patients with otherwise refractory disease. Despite these successes, extending CAR-T cell therapy to solid tumors remains challenging due to antigen heterogeneity, poor T cell infiltration, and the immunosuppressive tumor microenvironment (TME). Beyond oncology, CAR-T cell therapy has also shown promise in autoimmune diseases, where early clinical studies suggest that B cell-directed CAR-T cells can induce sustained remission in conditions such as systemic lupus erythematosus. This review highlights advances in CAR-T cell engineering, including DNA- and mRNA-based platforms for ex vivo and in vivo programming, and discusses emerging strategies to enhance CAR-T cell trafficking, persistence, and resistance to TME. Full article

Mutations in TP53 inhibit p53 protective behaviors including cell cycle arrest, DNA damage repair protein recruitment, and apoptosis. The ubiquity of p53 in genome-stabilizing functions leads to an aberrant tumor microenvironment in TP53-mutated myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Profound immunosuppression mediated by myeloid-derived suppressor cells, the upregulation of cytokines and cell-surface receptors on leukemic cells, the suppression of native immune regulator cells, and metabolic aberrations in the bone marrow are features of the TP53-mutated AML/MDS marrow microenvironment. These localized changes in the bone marrow microenvironment (BMME) explain why traditional therapies for MDS/AML, including chemotherapeutics and hypomethylating agents, are not as effective in TP53-mutated myeloid neoplasms and demonstrate the dire need for new treatments in this patient population. The unique pathophysiology of TP53-mutated disease also provides new therapeutic approaches which are being studied, including intracellular targets (MDM2, p53), cell-surface protein biologics (immune checkpoint inhibitors, BiTE therapy, and antibody–drug conjugates), cell therapies (CAR-T, NK-cell), signal transduction pathways (Hedgehog, Wnt, NF-κB, CCRL2, and HIF-1α), and co-opted biologic pathways (cholesterol synthesis and glycolysis). In this review, we will discuss the pathophysiologic anomalies of the tumor microenvironment in TP53-mutant MDS/AML, the hypothesized mechanisms of chemoresistance it imparts, and how novel therapies are leveraging diverse therapeutic targets to address this critical area of need. Full article

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of relapsed or refractory (r/r) diffuse large B-cell lymphoma (DLBCL); however, a significant proportion of patients fail to achieve a durable response, underscoring the need for reliable predictive biomarkers. We characterize T-lymphocyte subpopulations in apheresis samples from 23 r/r large B-cell lymphoma (LBCL) patients who received axicabtagene ciloleucel (axi-cel) to identify pre-treatment cell biomarkers associated with CAR T-cell kinetics and clinical outcomes. Immunophenotyping of T-cells within fresh apheresis samples and monitoring of circulating CAR T-cells were performed by multiparametric flow cytometry. The median peak CAR T-cell count was 45.2 CAR T-cells/mL. Strong CAR-T expanders (≥45.2 CAR T-cells/mL) exhibited higher values of both CD4⁺ (p = 0.011) and CD8⁺ (p = 0.023) central memory T-cells (T_CM; CCR7⁺CD45RA⁻), as well as lower proportions of CD8⁺CD38⁺ T-cells in apheresis samples. In apheresis, a cut-off value of >4.3% of CD8⁺ T_CM predicted strong CAR-T expansion (AUC: 0.80; p = 0.023) and superior progression-free survival (p = 0.04) compared with patients who had CD8⁺ T_CM below the cut-off. Our data suggest that high frequencies of CD8⁺ T_CM cells in apheresis samples may represent a promising pre-treatment biomarker associated with strong CAR-T expansion and superior clinical outcome in r/r LBCL patients following axi-cel. Full article

Multiple myeloma (MM) is a challenging hematologic malignancy characterized by clonal plasma cell proliferation, often leading to significant morbidity and mortality worldwide. Despite advances in chemotherapy and CAR-T therapies, MM remains incurable due to tumor heterogeneity, immune evasion, and microenvironment remodeling—exacerbated by toxicities like cytokine release syndrome and myelosuppression. This urgent unmet need demands innovative strategies. In this review, we assess cutting-edge RNA-based therapeutics for MM modulation, drawing on preclinical and clinical evidence on modalities including mRNA vaccines, small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs), and microRNA (miRNA) mimics/inhibitors. We further explore RNA-engineered cell therapies, such as transient CAR-T platforms and lipid nanoparticle-delivered systems targeting the bone marrow niche. By integrating these insights, we underscore RNA technologies’ transformative potential to achieve durable remissions, overcome resistance, and reduce costs—paving the way for personalized, safer treatments in refractory MM. Full article

Human immunodeficiency virus type 1 (HIV-1) continues to be a major global health burden. Combination antiretroviral therapy (cART) effectively abrogates HIV-1 replication and has transformed HIV-1 infection from a fatal to chronic disease. While ART can suppress viremia to undetectable levels in people living with HIV-1 (PWH), a small reservoir of cells infected with replication-competent HIV-1 persists and can lead to viral rebound upon ART interruption. This persistent HIV-1 reservoir can be quantified and characterized by measuring replication of infectious HIV-1 using a quantitative viral outgrowth assay (qVOA), or by measuring HIV-1 DNA, RNA, or protein levels as a proxy for the reservoir. Tools to quantify the reservoir in these distinct molecular compartments have been developed for HIV-1 subtype B, which is predominant in the Global North. However, non-B subtypes constitute the majority of HIV-1 infections worldwide. Here, we discuss the wide range of reservoir quantitation and characterization tools, explore their limitations, and, where applicable, their adaptations to non-B subtypes. We conclude that standardized tools should be used to characterize reservoir dynamics of HIV-1 B and non-B subtypes. These tests should be well-validated and accessible to all laboratories world-wide to be able to draw conclusions about subtype-specific reservoir dynamics. Full article

Prostate cancer (PCa) is the leading cause of cancer-related deaths worldwide and the second most common cancer among men. Treatment options depend on factors like age, androgen sensitivity, PSA levels, Gleason score, TNM stage, and recurrence risk. Available treatments include hormonal therapy, radiation, surgery, and chemotherapy. Early immunological treatments were limited by poor lymphocyte infiltration and an immunosuppressive environment. Today, strategies such as dendritic cell vaccines, immune checkpoint inhibitors (ICIs), and adoptive cell therapy (ACT) are used. ACT, especially CAR T-cell strategies, aims to overcome traditional treatment limitations, particularly in advanced and metastatic castration-resistant prostate cancer (mCRPC), though it remains in early development. Personalized medicine uses molecular insights from the diseased tissue to tailor treatments. Variability in patient response, due to tumor heterogeneity and prior treatments, highlights the importance of personalized and combination therapies as future strategies for effective immunotherapy. This review explores the current landscape of PCa. We analyze treatment guidelines established by NCCN and EANM-ESTRO-ESUR-ISUP-SIOG. We comprehensively examine immunotherapeutic strategies currently available or under investigation for prostate cancer, with particular emphasis on ICIs, ACT with a focus on CAR T-cell therapy, combination approaches and therapeutic synergies, and predictive biomarkers of immunotherapy response. Additionally, we discuss the challenges and future directions in the implementation of immunotherapy for the management of prostate cancer. Full article

Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary approach in immunotherapy that has shown remarkable success in the treatment of blood cancers. Many preclinical studies are currently underway worldwide to extend the CAR-T-cell therapy benefits to a broad spectrum of cancers, using rodent models. Alternative in vivo platforms are essential for overcoming the drawbacks associated with rodent models, including immunodeficiency in humanized models, ethical concerns, extended time requirements, and cost. In this work, we used the chicken chorioallantoic membrane (CAM) assay to evaluate the in vivo efficacy of cluster-of-differentiation 19 (CD19)-targeting CAR-T cells expressing a second-generation CAR construct against human lymphoma derived from the Raji cell line. Our results confirm the efficacy of selected CAR-T cells on tumor growth, metastasis, and angiogenesis. Further, the chicken embryo has an intrinsic active immune system. Therefore, the dialog between CAR-T cells and endogenous immune cells, as well as their participation in the tumor challenge, has also been studied. In conclusion, our study demonstrates that the chicken CAM assay provides a relevant in vivo, 3Rs (Replacement, Reduction and Refinement)-compliant new approach methodology (NAM), which is well-suited for the current needs of preclinical research on CAR-T-cell therapy. Full article

Lentiviral transduction remains the gold standard in adoptive modified cellular therapy, such as CAR-T; however, genome integration is not always desirable, such as when treating non-fatal autoimmune disease or for additional editing steps using CRISPR to produce allogeneic CAR-modified cells. Delivering in vitro-transcribed (IVT) mRNA represents an alternative solution but the labile nature of mRNA has led to efforts to improve half-life and translation efficiencies using a range of approaches including chemical and structural modifications. In this study, we explore the role of N⁶–methyladenosine (m6A) in a CD19-CAR sequence when delivered to T cells as an IVT mRNA. In silico analysis predicted the presence of four m6A consensus (DRACH) motifs in the CAR coding sequence and treating T cells with an inhibitor of the m6A methyltransferase (METTL3) resulted in a significant reduction in CAR protein expression. RNA analysis confirmed m6A bases at three of the predicted sites, indicating that the modification occurs independently of nuclear transcription. Synonymous mutation of the DRACH sites reduced the levels of CAR protein from 15 to >50% depending on the T cell donor. We also tested a panel of CAR transcripts with different UTRs, some containing m6A consensus motifs, and identified those which further improved protein expression. Furthermore, we found that the methylation of consensus m6A sites seems to be somewhat sequence-context-dependent. These findings demonstrate the importance of the m6A modification in stabilising and enhancing expression from IVT-derived mRNA and that this occurs within the cell, meaning targeted in vitro chemical modification during mRNA manufacturing may not be necessary. Full article

Systemic sclerosis (SSc) is a disease in which malfunctioning immune cells lead to the formation of autoantibodies that damage blood vessels and body tissues. Fibrosis then develops in the affected organs. Its complex pathogenesis involves multiple immune and stromal cell types, soluble mediators, and dysregulated tissue repair, resulting in heterogeneous clinical manifestations and poor prognosis. Current disease-modifying therapies provide only modest benefits, often slowing but rarely reversing disease progression, and are associated with considerable adverse effects. These limitations have spurred the development of cell-based therapeutic strategies aimed at restoring immune tolerance and promoting tissue repair. In this review, we summarize recent advances in hematopoietic stem cell transplantation, mesenchymal stem cell therapy, and adoptive regulatory T cell transfer and highlight the emerging role of chimeric antigen receptor (CAR)-T cell therapy as a transformative approach for SSc. Collectively, these evolving strategies hold the potential to improve survival, achieve durable remissions, and significantly enhance quality of life for patients with SSc. Full article

B-cells and plasmablasts have emerged as central organizers of autoimmune pathogenesis, extending far beyond their classical role as antibody-producing cells to orchestrate immune circuits, tissue microenvironments, and therapeutic trajectories. Advances in single-cell technologies, high-dimensional cytometry, and B-cell receptor sequencing have uncovered a dynamic continuum of B-cell differentiation programs that drive clinical heterogeneity across systemic autoimmune diseases. Plasmablasts, in particular, have gained recognition as highly responsive sensors of immune activation: they expand during flares, encode interferon-driven and extrafollicular responses, and correlate with disease severity. Autoantibody profiles, long viewed as static diagnostic signatures, are now understood as durable molecular footprints of distinct B-cell pathways. In this review, we propose an endotype-based framework integrating B-cell circuits with clinical phenotypes, illustrate therapeutic decision-making through mechanistic case vignettes, and outline future strategies combining immunomonitoring, multi-omics, and precision therapeutics. We further address translational challenges and discuss complementary approaches, including T-cell modulation, FcRn inhibition, and antigen-specific tolerization. Full article

Background/Objectives: Chimeric antigen receptor (CAR) T cells are a powerful cancer therapy, but their function depends heavily on internal signaling domains and metabolic adaptability. Most studies evaluate CAR behavior upon antigen exposure, yet intrinsic signaling properties may pre-program CAR T cell states even in the absence of stimulation. This study investigates how CAR design and metabolic support shape baseline transcriptional programs, focusing on tonic signaling and NF-κB-related pathways. Methods: We engineered CAR T cells targeting HER2 or GPC3 antigens, incorporating either 4-1BB or CD28 co-stimulatory domains, respectively. A subset of cells was further modified with adenosine deaminase 1 (ADA1) and CD26 to degrade extracellular adenosine and supply inosine, a metabolic strategy termed metabolic refueling (MR). Bulk RNA-seq was performed on resting T cells without antigen stimulation. We analyzed differential gene expression, gene set enrichment (GO, KEGG, Hallmarks), and transcription factor activity (DoRothEA) to assess the impact of CAR design and MR on T cell programming. Results: All CAR T cells exhibited activation of NF-κB–centered inflammatory programs at baseline, indicating tonic signaling. GPC3 CAR T cells showed stronger baseline activation than HER2 CAR T cells. Metabolic refueling amplified these programs without altering their directionality, enhancing inflammatory, survival, and effector modules. Transcription factor activity scores mirrored these trends, highlighting RELA, FOS, and STATs as key regulatory nodes. Conclusions: CAR-intrinsic features, notably co-stimulatory domain choice, define the tonic NF-κB activation tone in resting CAR T cells. Metabolic refueling boosts these baseline states without overstimulation, suggesting it may be especially valuable for weaker CAR constructs. These findings provide a framework for tuning CAR T cell function through combinatorial design strategies targeting signaling and metabolism. Full article

The manufacturing process contributes significantly to the proliferation, metabolic state, and functional persistence of chimeric antigen receptor (CAR)-T cells. However, how different culture systems regulate CAR-T cell metabolism and thereby influence their long-term antitumor activity remains poorly understood. In this study, we compared dynamic cultivation using a wave bioreactor with static expansion systems (gas-permeable and conventional T-flasks) for the production of CD99-specific CAR-T cells. CAR-T cells expanded by the wave bioreactor exhibited faster proliferation and stronger cytotoxicity during culture. Upon repeated antigen stimulation, they retained these enhanced functional properties and showed the reduced expression of immune checkpoint molecules, preferentially preserved memory-like subsets, and displayed transcriptional features consistent with memory maintenance and exhaustion resistance. Targeted metabolomic profiling revealed enhanced Tricarboxylic Acid (TCA) cycle activity and features consistent with sustained oxidative phosphorylation, supporting mitochondrial-centered metabolic reprogramming. In a Ewing sarcoma xenograft model, wave bioreactor-cultured CAR-T cells showed a greater percentage of memory-like tumor-infiltrating lymphocytes. Collectively, these results indicate that wave bioreactor-based dynamic cultivation promotes mitochondrial metabolic reprogramming, which is characterized by an enhanced TCA cycle and sustained oxidative phosphorylation, thereby sustaining CAR-T cell functionality and providing a robust platform for the manufacturing of potent and durable cellular therapeutics. Full article

Multiple sclerosis (MS) is a chronic, heterogeneous, multifactorial, immune-mediated neurodegenerative disease of the central nervous system that affects the working-age population. Its development is influenced by both genetic and environmental factors. A pathological hallmark of MS is the formation of demyelinating lesions in the brain and spinal cord, which are associated with neuronal damage caused by autoaggressive immune factors (T cells, B cells, and myeloid cells). Focal lesions are believed to be caused by the infiltration of immune cells into the central nervous system (CNS) parenchyma with concomitant tissue damage. Multiple sclerosis represents a significant social problem due to the high cost of available treatments, as well as the deterioration of employment prospects and job retention for both patients and their caregivers. Advances in MS diagnostic methods have enabled disease detection at early stages and correction of immune response impairments. Concurrently, treatments for MS patients are actively being studied, with the ongoing development of novel methods for targeted and cellular immunotherapy. This review primarily discusses approaches to cellular immunotherapy and methods of influencing the cellular arm of immunopathogenesis in multiple sclerosis. Full article

The stable and safe integration of exogenous DNA into the genome is crucial to both genetic engineering and gene therapy. Traditional transgenesis approaches, such as those using retroviral vectors, result in random genomic integration, posing the risk of insertional mutagenesis and transcriptional dysregulation. Safe harbor sites (SHSs), genomic loci that support reliable transgene expression without compromising endogenous gene function, genomic integrity, or cellular physiology, have been identified and characterized across various model organisms. Well-established SHSs such as AAVS1, ROSA26, and CLYBL are routinely utilized for targeted transgene integration in human cells. Recent advances in genome architecture, gene regulation, and genome editing technologies are driving the discovery of novel SHSs for precise and safe genetic modification. This review aims to provide a comprehensive overview of SHSs and their applications that will guide investigators in the choice of SHS, especially when complementary sites are needed for more than one transgene integration. First, it outlines safety and functional criteria that qualify a genomic site as a safe harbor site. It then discusses the two primary strategies for identifying SHSs: i) traditional lentiviral-based random transgenesis, and ii) modern genome-wide in silico screening followed by CRISPR-based validation. This review also provides an updated catalogue of currently known SHSs in the human genome, detailing their characteristics, uses, and limitations. Additionally, it discusses the diverse applications of SHSs in basic research, gene therapy, CAR T cell-based therapy, and biotechnological production systems. Finally, it concludes by highlighting challenges in identifying universally applicable SHSs and outlines future directions for their refinement and validation across biological systems. Full article