Tumor-Derived Extracellular Vesicles Impair CD171-Specific CD4+ CAR T Cell Efficacy.

Chimeric antigen receptor (CAR) T cell efficacy against solid tumors is currently limited by several immune escape mechanisms, which may include tumor-derived extracellular vesicles. Advanced neuroblastoma is an aggressive childhood tumor without curative treatment options for most relapsed patients today. We here evaluated the role of tumor-derived extracellular vesicles on the efficacy of CAR T cells targeting the neuroblastoma-specific antigen, CD171. For this purpose, CAR T cell activation, cytokine production, exhaustion, and tumor cell-directed cytotoxicity upon co-culture was evaluated. Tumor-derived extracellular vesicles isolated from SH-SY5Y neuroblastoma cells neither affected CAR T cell activation nor expression of inhibitory markers. Importantly, exposure of CD4+ CD171-specific CAR T cells to tumor-derived extracellular vesicles significantly impaired tumor cytotoxicity of CAR T cells. This effect was independent of neurotrophic receptor tyrosine kinases 1 or 2 (NTRK1, NTRK2) expression, which is known to impact immune responses against neuroblastoma. Our results demonstrate for the first time the impact of tumor-derived extracellular vesicles and non-cell-mediated tumor-suppressive effects on CD4+ CAR T cell efficacy in a preclinical setting. We conclude that these factors should be considered for any CAR T cell-based therapy to make CAR T cell therapy successful against solid tumors.

Central memory phenotype drives success of checkpoint inhibition in combination with CAR T cells.

The immunosuppressive microenvironment in solid tumors is thought to form a barrier to the entry and efficacy of cell-based therapies such as chimeric antigen receptor (CAR) T cells. Combining CAR T cell therapy with checkpoint inhibitors has been demonstrated to oppose immune escape mechanisms in solid tumors and augment antitumor efficacy. We evaluated PD-1/PD-L1 signaling capacity and the impact of an inhibitor of this checkpoint axis in an in vitro system for cancer cell challenge, the coculture of L1CAM-specific CAR T cells with neuroblastoma cell lines. Fluorescence-activated cell sorting-based analyses and luciferase reporter assays were used to assess PD-1/PD-L1 expression on CAR T and tumor cells as well as CAR T cell ability to kill neuroblastoma cells. Coculturing neuroblastoma cell lines with L1CAM-CAR T cells upregulated PD-L1 expression on neuroblastoma cells, confirming adaptive immune resistance. Exposure to neuroblastoma cells also upregulated the expression of the PD-1/PD-L1 axis in CAR T cells. The checkpoint inhibitor, nivolumab, enhanced L1CAM-CAR T cell-directed killing. However, nivolumab-enhanced L1CAM-CAR T cell killing did not strictly correlate with PD-L1 expression on neuroblastoma cells. In fact, checkpoint inhibitor success relied on strong PD-1/PD-L1 axis expression in the CAR T cells, which in turn depended on costimulatory domains within the CAR construct, and more importantly, on the subset of T cells selected for CAR T cell generation. Thus, T cell subset selection for CAR T cell generation and CAR T cell prescreening for PD-1/PD-L1 expression could help determine when combination therapy with checkpoint inhibitors could improve treatment efficacy.

Applications of high-resolution magic angle spinning MRS in biomedical studies I-cell line and animal models.

High-resolution magic angle spinning (HRMAS) MRS allows for direct measurements of non-liquid tissue and cell specimens to present valuable insights into the cellular metabolisms of physiological and pathological processes. HRMAS produces high-resolution spectra comparable to those obtained from solutions of specimen extracts but without complex metabolite extraction processes, and preserves the tissue cellular structure in a form suitable for pathological examinations following spectroscopic analysis. The technique has been applied in a wide variety of biomedical and biochemical studies and become one of the major platforms of metabolomic studies. By quantifying single metabolites, metabolite ratios, or metabolic profiles in their entirety, HRMAS presents promising possibilities for diagnosis and prediction of clinical outcomes for various diseases, as well as deciphering of metabolic changes resulting from drug therapies or xenobiotic interactions. In this review, we evaluate HRMAS MRS results on animal models and cell lines reported in the literature, and present the diverse applications of the method for the understanding of pathological processes and the effectiveness of therapies, development of disease animal models, and new progress in HRMAS methodology.