Background/Objectives: Natural killer (NK) cells play a crucial role in tumor surveillance by exerting cytotoxic activity and modulating immune responses. However, tumors employ diverse evasion strategies that limit NK cell effectiveness. This review aims to explore the molecular mechanisms of NK cell activation and inhibition in cancer, the influence of the tumor microenvironment, and the latest advancements in NK cell-based immunotherapies, including adoptive NK cell transfer and Chimeric Antigen Receptor-Natural Killer (CAR-NK) cell therapies. Methods: A comprehensive literature review was conducted, prioritizing peer-reviewed studies from the last decade on NK cell biology, tumor immune evasion, and immunotherapeutic applications. The analysis includes data from preclinical models and clinical trials evaluating NK cell expansion strategies, cytokine-based stimulation, and CAR-NK cell therapy developments. Results: NK cells eliminate tumors through cytotoxic granule release, death receptor pathways, and cytokine secretion. However, tumor cells evade NK-mediated immunity by downregulating activating ligands, secreting immunosuppressive molecules, and altering the tumor microenvironment. Novel NK cell-based therapies, such as CAR-NK cells and combination approaches with immune checkpoint inhibitors, enhance NK cell persistence and therapeutic efficacy against both hematologic and solid malignancies. Clinical trials suggest improved safety profiles compared to CAR-T therapies, with reduced cytokine release syndrome and graft-versus-host disease. Conclusions: While NK cell-based immunotherapies hold great promise, challenges remain, including limited persistence and tumor-induced immunosuppression. Addressing these hurdles will be critical for optimizing NK cell therapies and advancing next-generation, off-the-shelf immunotherapeutics for broader clinical applications.

Monitoring protein kinases in their native environment is essential for understanding cellular signaling. This study introduces triangulenium‐based peptide biosensors for detection of the activity of two key protein kinases involved in T cell immune response: Akt1 and Src. The substrate biosensors incorporate a long‐lifetime fluorophore from the triangulenium dye family, which undergoes quenching by photoinduced electron transfer (PET) from different amino acid side groups. Upon kinase‐mediated phosphorylation, the PET quenching is significantly suppressed, increasing both fluorescence intensity and fluorescence lifetime (τ _FL ). This lifetime extension (up to 15 ns) enables the use of fluorescence lifetime imaging microscopy (FLIM) and time‐gated filtering to effectively eliminate cellular autofluorescence. In vitro and in extracto assays confirmed the biosensors' high affinity and specificity for endogenous kinases. FLIM experiments in HeLa cells demonstrated efficient uptake, biocompatibility, and quantitative reporting of endogenous Src and Akt1 activities through robust, lifetime‑based responses selectively modulated by kinase activators and inhibitors. Crucially, the Akt1 biosensor tracked dynamic signaling changes in isolated primary T cells following T cell receptor stimulation using confocal microscopy and flow cytometry. This integrated framework provides a robust, non‐invasive platform for quantitative kinase analysis in living cells, with significant implications for immunological profiling and cancer research.

Dengue virus (DENV) is the most prevalent mosquito-borne viral disease with over ten million cases worldwide. There are four antigenically distinct, co-circulating DENV serotypes (DENV 1-4) capable of infecting humans. Given the lack of immunocompetent animal models and the limitations of known cell culture models, more physiologically relevant experimental models are needed to recapitulate DENV infection and host immune responses. Building on previous observations that DENV-2 and DENV-4 serotypes elicit distinct innate immune responses in monocyte-derived dendritic cells (moDC) in vitro , we utilized a human tonsil histoculture (HC) model to further investigate serotype-specific differences within the physiologically relevant human lymphoid environment. We show that human tonsil HCs preserved their tissue cytoarchitecture for up to 6 days in culture, including maintaining functional germinal centers and diverse immune populations within T and B cell compartments. Exposure of tonsil HCs to DENV-2 and DENV-4 showed that DENV-4 replication peaked earlier and induced enhanced innate immune activation compared to DENV-2, consistent with previous observations in human DCs. Moreover, by leveraging the structural and cellular complexity of the HC system, we further identified that DENV E protein co-localized with HLA-DR+ antigen-presenting cells, confirming that DENV-infected cells within tonsil HCs predominantly express antigen-presenting cell markers. Altogether, these findings demonstrate that different DENV serotypes can exhibit different viral replication dynamics and induce distinct immune responses within human lymphoid tissue. This establishes human tonsil HCs as human model system with intact cytoarchitecture that closely mirrors lymph node structure and function, providing a powerful platform to study antigen-driven and virus-specific human immune responses and ultimately to evaluate vaccine candidates and antiviral therapeutics.