Periodic Reporting for period 2 - ImmunoCode (Digital Single Cell Immunology: Decoding Cellular Interactions for Improved Immunotherapy)
Okres sprawozdawczy: 2020-05-01 do 2021-10-31
Successful immunotherapy against cancer is the result of a multitude of cellular interactions within the immune system. It is highly exciting how vaccination with small numbers of human plasmacytoid dendritic cells can induce anti-tumour immunity in metastatic cancer patients. Remarkably, how plasmacytoid dendritic cells, as an extremely rare subset of cells, can act as Swiss army knives to regulate the tight balance between tolerance and immunity: i.e. secrete massive amounts of type I interferons, prime T cells and exert cytotoxic effector functions, is still elusive. Do plasmacytoid dendritic cells possess all functions or comprises the population distinct functional subsets all with their specialized roles. This project will unravel which plasmacytoid dendritic cell is superior in inducing cytotoxic T cells for augmented cancer immunity. Despite advances in single cell technologies, understanding the role of heterogeneity in immune cell populations, remains poorly understood. In ImmunoCode, a novel and ambitious single cell technology platform will be developed, wherein innovative microfluidic approaches and single cell transcriptomics allow 1) functional analysis of single (pairs of) immune cells and 2) design of minimal environments under the omission of external factors that could influence cellular behaviour.
The main objectives of this project are: 1) understanding plasmacytoid dendritic cell heterogeneity, 2) generation of a microfluidic toolbox for high-throughput studies aimed at bi-directional crosstalk between pairs of single cells, 3) generate complex artificial microenvironments to assess the behaviour of single plasmacytoid dendritic cells in response to e.g. soluble messengers.
This approach will yield the unique opportunity to unravel plasmacytoid dendritic cell function and plasticity. Ultimately, the results generated in this project can have a great impact by refining the design of vaccine strategies and the development of differently composed cellular vaccines to battle cancer and infectious and auto-immune diseases.
The main objectives of this project are: 1) understanding plasmacytoid dendritic cell heterogeneity, 2) generation of a microfluidic toolbox for high-throughput studies aimed at bi-directional crosstalk between pairs of single cells, 3) generate complex artificial microenvironments to assess the behaviour of single plasmacytoid dendritic cells in response to e.g. soluble messengers.
This approach will yield the unique opportunity to unravel plasmacytoid dendritic cell function and plasticity. Ultimately, the results generated in this project can have a great impact by refining the design of vaccine strategies and the development of differently composed cellular vaccines to battle cancer and infectious and auto-immune diseases.
A droplet-based microfluidic platform has been developed and employed to dissect both phenotypic as well as functional heterogeneity in the plasmacytoid dendritic cell compartment. With this platform we have revealed the existence of multi-layered interferon responses ignited by so-called first responder cells. Moreover, scripts were generated to allow for automated, longitudinal and high-throughput screening of single cell response to soluble stimuli as well as between pairs of single cells. Currently, work is in progress to further develop the microfluidic platform to allow for measuring longitudinal single cell cytokine secretion in real-time. Furthermore, to understand the ability of single cells to generate immune responses we probed their behaviour in health and disease. A valve-based two-layer microfluidic cell (co-)culture system for temporal analysis of cellular signaling at single-cell level has been designed and optimized. Biological experiments with fibroblast reporter cell lines to understand the dynamics and control of type I interferon signalling have been performed. Multicellular valve-based two-layer microfluidic system has been designed for pulsatile cytokine delivery to cells in order to probe cell activation thresholds. We have performed numerous experiments with the abovementioned reporter cells to probe signal integration leading to single cell communication
For WP-1, in the upcoming period, a major action will be to focus on the role for plasmacytoid dendritic cells to activate cytotoxic T cells. With this systems immunology approach we can study immune interactions at the level of individual plasmacytoid dendritic cells which is the only way to unambiguously elucidate which cellular properties of plasmacytoid dendritic cells correlate with an effective T cell induction with desired anti-cancer properties. Moreover, we will continue with monitoring cellular responses longitudinally, high-throughput and in a multiplexed and quantifiable fashion to revolutionize our understanding of cellular interactions. Interactions with academic and industrial stakeholders will increase and strengthen the in this project generated technological advances to further ensure the translation of results to improved understanding of cellular responses in auto-immune patients as well as improved clinical products for immunotherapy. Finally we will continue to optimize and expand our microfluidic toolbox to enable the generation of biological experiments that currently are difficult or virtually impossible to perform.
The regulation of immune responses demands complex decision-making processes, implemented by a sophisticated network of cells and molecules that interact cooperatively to generate functional responses. The microenvironment plays a crucial role in immunological decision making and orchestrates the behaviour of cells. For WP-2, the expected progress in the upcoming period will be the execution of experiments that allow to obtain insight in the regulation of type I interferon responses by first and second responder cells. Results will highlight the value of high-throughput quantitative measurements at the single-cell level to reveal how biological systems operate. Understanding cellular activation thresholds may lead to new and improved therapeutic targets specifically aimed at boosting or disrupting plasmacytoid dendritic cell-derived type I interferon responses. Furthermore, together with our collaborators the ongoing mathematical modelling and combinatorial perturbation profiling will provide insight how immunological circuits are constructed and how paracrine signalling by soluble factors present in microenvironments affects cellular activation thresholds in the generation of type I interferon responses.
The regulation of immune responses demands complex decision-making processes, implemented by a sophisticated network of cells and molecules that interact cooperatively to generate functional responses. The microenvironment plays a crucial role in immunological decision making and orchestrates the behaviour of cells. For WP-2, the expected progress in the upcoming period will be the execution of experiments that allow to obtain insight in the regulation of type I interferon responses by first and second responder cells. Results will highlight the value of high-throughput quantitative measurements at the single-cell level to reveal how biological systems operate. Understanding cellular activation thresholds may lead to new and improved therapeutic targets specifically aimed at boosting or disrupting plasmacytoid dendritic cell-derived type I interferon responses. Furthermore, together with our collaborators the ongoing mathematical modelling and combinatorial perturbation profiling will provide insight how immunological circuits are constructed and how paracrine signalling by soluble factors present in microenvironments affects cellular activation thresholds in the generation of type I interferon responses.