At the interface between the innate and adaptive immune system, dendritic cells (DCs) play key roles in tumour immunity and hold a hitherto unrealized potential for cancer immunotherapy. Here we ...review the role of distinct DC subsets in the tumour microenvironment, with special emphasis on conventional type 1 DCs. Integrating new knowledge of DC biology and advancements in cell engineering, we provide a blueprint for the rational design of optimized DC vaccines for personalized cancer medicine.
The tumor microenvironment encompasses an intertwined ensemble of both transformed cancer cells and non-transformed host cells, which together establish a signaling network that regulates tumor ...progression. By conveying both homo- and heterotypic cell-to-cell communication cues, tumor-derived extracellular vesicles (tEVs) modulate several cancer-associated processes, such as immunosuppression, angiogenesis, invasion, and metastasis. Herein we discuss how recent methodological advances in the isolation and characterization of tEVs may help to broaden our understanding of their functions in tumor biology and, potentially, establish their utility as cancer biomarkers.
Beltraminelli et al. review methodological advances in the isolation, analysis, and biological characterization of tumor-derived extracellular vesicles (tEVs), arguing for the field to embrace techniques that capture the complexity of tEVs within the tissue of origin.
This study introduces a T cell enrichment process, capitalizing on the size differences between activated and unactivated T cells to facilitate the isolation of activated, transducible T cells. By ...employing multidimensional double spiral (MDDS) inertial sorting, our approach aims to remove unactivated or not fully activated T cells post-activation, consequently enhancing the efficiency of chimeric antigen receptor (CAR) T cell manufacturing. Our findings reveal that incorporating a simple, label-free, and continuous MDDS sorting step yields a purer T cell population, exhibiting significantly enhanced viability and CAR-transducibility (with up to 85% removal of unactivated T cells and approximately 80% recovery of activated T cells); we found approximately 2-fold increase in CAR transduction efficiency for a specific sample, escalating from ∼10% to ∼20%, but this efficiency highly depends on the original T cell sample as MDDS sorting would be more effective for samples possessing a higher proportion of unactivated T cells. This new cell separation process could augment the efficiency, yield, and cost-effectiveness of CAR T cell manufacturing, potentially broadening the accessibility of this transformative therapy and contributing to improved patient outcomes.
Interleukin-2 is a pleiotropic cytokine that mediates both pro- and anti-inflammatory functions. Immune cells naturally differ in their sensitivity to IL-2 due to cell type and activation ...state-dependent expression of receptors and signaling pathway components. To probe differences in IL-2 signaling across cell types, we used structure-based design to create and profile a series of IL-2 variants with the capacity to titrate maximum signal strength in fine increments. One of these partial agonists, IL-2-REH, specifically expanded Foxp3+ regulatory T cells with reduced activity on CD8+ T cells due to cell type-intrinsic differences in IL-2 signaling. IL-2-REH elicited cell type-dependent differences in gene expression and provided mixed therapeutic results: showing benefit in the in vivo mouse dextran sulfate sodium (DSS) model of colitis, but no therapeutic efficacy in a transfer colitis model. Our findings show that cytokine partial agonists can be used to calibrate intrinsic differences in response thresholds across responding cell types to narrow pleiotropic actions, which may be generalizable to other cytokine and growth factor systems.
Biomaterial properties that modulate T cell activation, growth, and differentiation are of significant interest in the field of cellular immunotherapy manufacturing. In this work, a new platform ...technology that allows for the modulation of various activation particle design parameters important for polyclonal T cell activation is presented. Artificial antigen presenting cells (aAPCs) are successfully created using supported lipid bilayers on various cell‐templated silica microparticles with defined membrane fluidity and stimulating antibody density. This panel of aAPCs is used to probe the importance of activation particle shape, size, membrane fluidity, and stimulation antibody density on T cell outgrowth and differentiation. All aAPC formulations are able to stimulate T cell growth, and preferentially promote CD8+ T cell growth over CD4+ T cell growth when compared to commercially available pendant antibody‐conjugated particles. T cells cultured with HeLa‐ and red blood cell–templated aAPCs have a less‐differentiated and less‐exhausted phenotype than those cultured with spherical aAPCs with matched membrane coatings when cultured for 14 days. These results support continued exploration of silica‐supported lipid bilayers as an aAPC platform.
Silica microparticles templated from monodisperse human cells are used to create supported lipid bilayers displaying T cell activating antibodies with varying size, shape, membrane fluidity, and antibody loading density. These particles promote polyclonal human T cell expansion and demonstrate that antibody density and particle shape impact T cell differentiation during expansion.
The immunostimulatory intracellular domains (ICDs) of chimaeric antigen receptors (CARs) are essential for converting antigen recognition into antitumoural function. Although there are many possible ...combinations of ICDs, almost all current CARs rely on combinations of CD3𝛇, CD28 and 4-1BB. Here we show that a barcoded library of 700,000 unique CD19-specific CARs with diverse ICDs cloned into lentiviral vectors and transduced into Jurkat T cells can be screened at high throughput via cell sorting and next-generation sequencing to optimize CAR signalling for antitumoural functions. By using this screening approach, we identified CARs with new ICD combinations that, compared with clinically available CARs, endowed human primary T cells with comparable tumour control in mice and with improved proliferation, persistence, exhaustion and cytotoxicity after tumour rechallenge in vitro. The screening strategy can be adapted to other disease models, cell types and selection conditions, and could be used to improve adoptive cell therapies and to expand their utility to new disease indications.
Biomaterial properties that modulate T cell activation, growth, and differentiation are of significant interest in the field of cellular immunotherapy manufacturing. In this work, we present a new ...platform technology that allows for the modulation of various activation particle design parameters important for polyclonal T cell activation. We successfully create artificial antigen presenting cells (aAPCs) using supported lipid bilayers on various cell-templated silica microparticles with defined membrane fluidity and stimulating antibody density. We use this panel of aAPCs to probe the importance of activation particle shape, size, membrane fluidity, and stimulation antibody density on T cell outgrowth and differentiation. All aAPC formulations were able to stimulate T cell growth, and preferentially promoted CD8
+
T cell growth over CD4
+
T cell growth when compared to commercially available pendant antibody-conjugated particles. T cells cultured with HeLa and red blood cell templated aAPCs had a less differentiated and less exhausted phenotype than those cultured with spherical aAPCs with matched membrane coatings when cultured for 14 days. These results support continued exploration of silica supported lipid bilayers as an artificial antigen presenting cell platform.
Silica microparticles templated from monodisperse human cells
were used to create supported lipid bilayers displaying T cell activating antibodies with varying size, shape, membrane fluidity, and antibody loading density. These particles promote polyclonal human T cell expansion and demonstrate that antibody density and particle shape impact T cell differentiation during expansion.
Enhancer elements in the human genome control how genes are expressed in specific cell types and harbor thousands of genetic variants that influence risk for common diseases
. Yet, we still do not ...know how enhancers regulate specific genes, and we lack general rules to predict enhancer-gene connections across cell types
. We developed an experimental approach, CRISPRi-FlowFISH, to perturb enhancers in the genome, and we applied it to test >3,500 potential enhancer-gene connections for 30 genes. We found that a simple activity-by-contact model substantially outperformed previous methods at predicting the complex connections in our CRISPR dataset. This activity-by-contact model allows us to construct genome-wide maps of enhancer-gene connections in a given cell type, on the basis of chromatin state measurements. Together, CRISPRi-FlowFISH and the activity-by-contact model provide a systematic approach to map and predict which enhancers regulate which genes, and will help to interpret the functions of the thousands of disease risk variants in the noncoding genome.
PDF
