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T cells are an essential part of the immune system with crucial roles in adaptive response and the maintenance of tissue homeostasis. Depending on their microenvironment, T cells can be differentiated into multiple states with distinct functions. This myriad of cellular activities have prompted the development of numerous smart probes, ranging from small molecule fluorophores to nanoconstructs with variable molecular architectures and fluorescence emission mechanisms. In this Tutorial Review, we summarize recent efforts in the design, synthesis and application of smart probes for imaging T cells in tumors and inflammation sites by targeting metabolic and enzymatic biomarkers as well as specific surface receptors. Finally, we briefly review current strategies for how smart probes are employed to monitor the response of T cells to anti-cancer immunotherapies. We hope that this Review may help chemists, biologists and immunologists to design the next generation of molecular imaging probes for T cells and anti-cancer immunotherapies. [ABSTRACT FROM AUTHOR]

Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near‐infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity‐based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild‐type and GzmA knock‐out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen‐driven recognition of cancer cells in real time. [ABSTRACT FROM AUTHOR]

Increased levels of tumor‐associated macrophages (TAMs) are indicators of poor prognosis in most cancers. Although antibodies and small molecules blocking the recruitment of macrophages to tumors are under evaluation as anticancer therapies, these strategies are not specific for macrophage subpopulations. Herein we report the first enzyme‐activatable chemokine conjugates for effective targeting of defined macrophage subsets in live tumors. Our constructs exploit the high expression of chemokine receptors (e.g. CCR2) and the activity of cysteine cathepsins in TAMs to target these cells selectively over other macrophages and immune cells (e.g. neutrophils, T cells, B cells). Furthermore, we demonstrate that cathepsin‐activatable chemokines are compatible with both fluorescent and therapeutic cargos, opening new avenues in the design of targeted theranostic probes for immune cells in the tumor microenvironment. [ABSTRACT FROM AUTHOR]

Immunotherapy promotes the attack of cancer cells by the immune system; however, it is difficult to detect early responses before changes in tumor size occur. Here, we report the rational design of a fluorogenic peptide able to detect picomolar concentrations of active granzyme B as a biomarker of immune-mediated anticancer action. Through a series of chemical iterations and molecular dynamics simulations, we synthesize a library of FRET peptides and identify probe H5 with an optimal fit into granzyme B. We demonstrate that probe H5 enables the real-time detection of T cell-mediated anticancer activity in mouse tumors and in tumors from lung cancer patients. Furthermore, we show image-based phenotypic screens, which reveal that the AKT kinase inhibitor AZD5363 shows immune-mediated anticancer activity. The reactivity of probe H5 may enable the monitoring of early responses to anticancer treatments using tissue biopsies. Granzyme B is found in activated T cells and can be used as a marker of T cell activation. Here, the authors generate a fluorescent probe that can detect Granzyme B levels in tumours, and has the potential to be used as a biomarker of response to immunotherapy. [ABSTRACT FROM AUTHOR]