Live Imaging of CAR T Cell Ca2+ Signals in Tumor Slices Using Confocal Microscopy.

The immune synapse is a key structure organizing T-cell activation against foreign entities, such as cancer cells expressing neoantigens. One crucial step in this activation cascade is the intracellular Ca2+ ([Ca2+]i) response that shapes T cells for proliferation, differentiation, and cytotoxicity. The development of calcium probes coupled to real-time fluorescence microscopy has allowed a close study of this phenomenon in vitro. Such systems have provided valuable insights on the consequences of Ca2+ responses on T cells, including cytotoxicity and cytoskeletal remodeling. However, in vitro models do not recapitulate the tissue architecture that T cells come in contact with in vivo. Thus, there is a growing necessity for better understanding the factors influencing Ca2+ response in T cells including in genetically modified T cells (e.g., CAR T cells). In this methodology chapter, we describe an experimental system to measure [Ca2+]i signals of CAR T cells loaded with the calcium probe Fluo-4 on fresh tumor slices. Combined with confocal fluorescent imaging, this model offers an approach to image early T-cell activation in a three-dimensional (3D) tissue environment.

CAR T-cell behavior and function revealed by real-time imaging.

Adoptive transfer of T-cells expressing chimeric antigen receptors (CAR) has shown remarkable clinical efficacy against advanced B-cell malignancies. Nonetheless, the field of CAR T-cells is currently facing several major challenges. In particular, the CAR T-cell strategy has not yet produced favorable clinical responses when targeting solid tumors. In this context, it is of paramount importance to understand the determinants that limit the efficacy of T-cell-based immunotherapy. Characterization of CAR T-cells is usually based on flow cytometry and whole-transcriptome profiling. These approaches have been very valuable to determine intrinsic elements that condition T-cell ability to proliferate and expand. However, they do not take into account spatial and kinetic aspects of T-cell responses. In particular, in order to control tumor growth, CAR T-cells need to enter into the tumor, migrate within a complex tumor environment, and form productive conjugates with their targets. Advanced imaging techniques combined with innovative preclinical models represent promising tools to uncover the dynamics of CAR T-cells. In this review, we will discuss recent results on the biology of engineered T-cells that have been obtained with real-time imaging microscopy. Important notions have emerged from these imaging-based studies, such as the multi-killing potential of CAR T-cells. Finally, we will highlight how imaging techniques combined with other tools can solve remaining unresolved questions in the field of engineered T-cells.

New insights into CAR T cell-mediated killing of tumor cells.

Adoptive transfer of T cells genetically engineered to express chimeric antigen receptors (CAR) has demonstrated striking efficacy for the treatment of several hematological malignancies, including B-cell lymphoma, leukemia, and multiple myeloma. However, CAR T-cell efficacy has been very limited in most solid tumors. In this context, it is of paramount importance to understand the determinants that condition CAR T-cell success versus failure. To control tumor growth, CAR T cells need to form conjugates with their targets via the assembly of an immunological synapse. Here, we review recent advances showing that the adhesion between CAR T cells and cancer cells from solid tumors strengthens over time in an IFNγ- and ICAM-1-dependent manner, resulting in CAR T cell-mediated killing. We discuss how these findings can be exploited to increase the efficacy of the CAR T-cell strategy against solid tumors.

CAR T cells targeting Aspergillus fumigatus are effective at treating invasive pulmonary aspergillosis in preclinical models.

Aspergillus fumigatus is a ubiquitous mold that can cause severe infections in immunocompromised patients, typically manifesting as invasive pulmonary aspergillosis (IPA). Adaptive and innate immune cells that respond to A. fumigatus are present in the endogenous repertoire of patients with IPA but are infrequent and cannot be consistently isolated and expanded for adoptive immunotherapy. Therefore, we gene-engineered A. fumigatus-specific chimeric antigen receptor (Af-CAR) T cells and demonstrate their ability to confer antifungal reactivity in preclinical models in vitro and in vivo. We generated a CAR targeting domain AB90-E8 that recognizes a conserved protein antigen in the cell wall of A. fumigatus hyphae. T cells expressing the Af-CAR recognized A. fumigatus strains and clinical isolates and exerted a direct antifungal effect against A. fumigatus hyphae. In particular, CD8+ Af-CAR T cells released perforin and granzyme B and damaged A. fumigatus hyphae. CD8+ and CD4+ Af-CAR T cells produced cytokines that activated macrophages to potentiate the antifungal effect. In an in vivo model of IPA in immunodeficient mice, CD8+ Af-CAR T cells localized to the site of infection, engaged innate immune cells, and reduced fungal burden in the lung. Adoptive transfer of CD8+ Af-CAR T cells conferred greater antifungal efficacy compared to CD4+ Af-CAR T cells and an improvement in overall survival. Together, our study illustrates the potential of gene-engineered T cells to treat aggressive infectious diseases that are difficult to control with conventional antimicrobial therapy and support the clinical development of Af-CAR T cell therapy to treat IPA.

Time to evolve: predicting engineered T cell-associated toxicity with next-generation models.

Despite promising clinical results in a small subset of malignancies, therapies based on engineered chimeric antigen receptor and T-cell receptor T cells are associated with serious adverse events, including cytokine release syndrome and neurotoxicity. These toxicities are sometimes so severe that they significantly hinder the implementation of this therapeutic strategy. For a long time, existing preclinical models failed to predict severe toxicities seen in human clinical trials after engineered T-cell infusion. However, in recent years, there has been a concerted effort to develop models, including humanized mouse models, which can better recapitulate toxicities observed in patients. The Accelerating Development and Improving Access to CAR and TCR-engineered T cell therapy (T2EVOLVE) consortium is a public-private partnership directed at accelerating the preclinical development and increasing access to engineered T-cell therapy for patients with cancer. A key ambition in T2EVOLVE is to design new models and tools with higher predictive value for clinical safety and efficacy, in order to improve and accelerate the selection of lead T-cell products for clinical translation. Herein, we review existing preclinical models that are used to test the safety of engineered T cells. We will also highlight limitations of these models and propose potential measures to improve them.

Time 2EVOLVE: predicting efficacy of engineered T-cells - how far is the bench from the bedside?

Immunotherapy with gene engineered CAR and TCR transgenic T-cells is a transformative treatment in cancer medicine. There is a rich pipeline with target antigens and sophisticated technologies that will enable establishing this novel treatment not only in rare hematological malignancies, but also in common solid tumors. The T2EVOLVE consortium is a public private partnership directed at accelerating the preclinical development of and increasing access to engineered T-cell immunotherapies for cancer patients. A key ambition in T2EVOLVE is to assess the currently available preclinical models for evaluating safety and efficacy of engineered T cell therapy and developing new models and test parameters with higher predictive value for clinical safety and efficacy in order to improve and accelerate the selection of lead T-cell products for clinical translation. Here, we review existing and emerging preclinical models that permit assessing CAR and TCR signaling and antigen binding, the access and function of engineered T-cells to primary and metastatic tumor ligands, as well as the impact of endogenous factors such as the host immune system and microbiome. Collectively, this review article presents a perspective on an accelerated translational development path that is based on innovative standardized preclinical test systems for CAR and TCR transgenic T-cell products.