Periodic Reporting for period 4 - ImmunoCode (Digital Single Cell Immunology: Decoding Cellular Interactions for Improved Immunotherapy)
Periodo di rendicontazione: 2023-05-01 al 2024-06-30
Successful cancer immunotherapy hinges on a complex web of cellular interactions within the immune system. It’s particularly exciting to see how vaccinations with small numbers of human plasmacytoid dendritic cells (pDCs) can trigger anti-tumor immunity in patients with metastatic cancer. Despite being a rare subset, pDCs function like the Swiss army knives of the immune system, balancing tolerance and immunity by secreting large amounts of type I interferons, priming T cells, and exerting cytotoxic functions. However, it remains unclear whether all pDCs possess these functions or if the population consists of distinct subsets with specialized roles. This project aimed to identify which pDCs are most effective at inducing cytotoxic T cells to enhance cancer immunity.
Despite advancements in single-cell technologies, the heterogeneity within immune cell populations is still not fully understood. The ImmunoCode project seeked to bridge this gap by developing an innovative single-cell technology platform. This platform would leverage cutting-edge microfluidic approaches and single-cell transcriptomics to enable functional analysis of individual immune cells and the creation of minimal environments free from external influences.
The main objectives of this project were:
1. Understanding the heterogeneity of plasmacytoid dendritic cells.
2. Developing a microfluidic toolbox for high-throughput studies of bi-directional crosstalk between pairs of single cells.
3. Creating complex artificial microenvironments to study the behavior of single pDCs in response to soluble messengers.
This approach would offer a unique opportunity to unravel the functions and plasticity of pDCs. Ultimately, the findings from this project could significantly impact the design of vaccine strategies and the development of diverse cellular vaccines to combat cancer, infectious diseases, and autoimmune disorders.
Final outcomes and impact
For a long-time, cellular communication was attributed to auto-, para-, endo-, or juxtacrine interactions. Only recently have a few studies proposed immune quorum sensing as a new mode of communication. Our platform revealed the presence and regulation of immune quorum sensing of IFN-I dynamics. The scientific impact here is significant, as our approach, technology, and methodologies are not restricted to specific proteins or cells but can be applied to virtually any cell type with expected heterogeneity or cytokine communication systems. This will provide a more comprehensive picture of how cytokine communication and cellular heterogeneity are intertwined, likely extending beyond current dogmas in understanding immune regulation.
Our approach is unique, as no other currently available technology, such as single cell RNA-seq, can study this exciting behavior of single cells. Understanding the molecular basis underlying the heterogeneity within IFN-I secreting cell systems will lead to new and better therapeutic strategies aimed at controlling type I IFN production.
The microfluidic technologies and analysis techniques developed and introduced in this project are certainly beyond the current state-of-the-art. The droplet-based microfluidic system that enables automated, high-throughput, real-time analysis of cytotoxic events is highly novel, as is the combined in-droplet cytokine detection assay. This innovative platform is a breakthrough technology with many possibilities for improving our understanding of cellular functional heterogeneity across a wide variety of immune cells. Consequently, the group of the Principal Investigator (PI) was regularly approached by both academic and industrial stakeholders to explore potential collaborative endeavors. The high level of interest from different stakeholders was unexpected but clearly indicates the potential high impact this technology could have across various fields.
All the objectives were met, leading to several new innovations published in high-impact journals.
Despite advancements in single-cell technologies, the heterogeneity within immune cell populations is still not fully understood. The ImmunoCode project seeked to bridge this gap by developing an innovative single-cell technology platform. This platform would leverage cutting-edge microfluidic approaches and single-cell transcriptomics to enable functional analysis of individual immune cells and the creation of minimal environments free from external influences.
The main objectives of this project were:
1. Understanding the heterogeneity of plasmacytoid dendritic cells.
2. Developing a microfluidic toolbox for high-throughput studies of bi-directional crosstalk between pairs of single cells.
3. Creating complex artificial microenvironments to study the behavior of single pDCs in response to soluble messengers.
This approach would offer a unique opportunity to unravel the functions and plasticity of pDCs. Ultimately, the findings from this project could significantly impact the design of vaccine strategies and the development of diverse cellular vaccines to combat cancer, infectious diseases, and autoimmune disorders.
Final outcomes and impact
For a long-time, cellular communication was attributed to auto-, para-, endo-, or juxtacrine interactions. Only recently have a few studies proposed immune quorum sensing as a new mode of communication. Our platform revealed the presence and regulation of immune quorum sensing of IFN-I dynamics. The scientific impact here is significant, as our approach, technology, and methodologies are not restricted to specific proteins or cells but can be applied to virtually any cell type with expected heterogeneity or cytokine communication systems. This will provide a more comprehensive picture of how cytokine communication and cellular heterogeneity are intertwined, likely extending beyond current dogmas in understanding immune regulation.
Our approach is unique, as no other currently available technology, such as single cell RNA-seq, can study this exciting behavior of single cells. Understanding the molecular basis underlying the heterogeneity within IFN-I secreting cell systems will lead to new and better therapeutic strategies aimed at controlling type I IFN production.
The microfluidic technologies and analysis techniques developed and introduced in this project are certainly beyond the current state-of-the-art. The droplet-based microfluidic system that enables automated, high-throughput, real-time analysis of cytotoxic events is highly novel, as is the combined in-droplet cytokine detection assay. This innovative platform is a breakthrough technology with many possibilities for improving our understanding of cellular functional heterogeneity across a wide variety of immune cells. Consequently, the group of the Principal Investigator (PI) was regularly approached by both academic and industrial stakeholders to explore potential collaborative endeavors. The high level of interest from different stakeholders was unexpected but clearly indicates the potential high impact this technology could have across various fields.
All the objectives were met, leading to several new innovations published in high-impact journals.
Main Results Achieved:
1. Uncovered multi-layered interferon responses.
2. Enabled automated, high-throughput single-cell screening.
3. Provided precise temporal analysis of cellular signaling.
4. Advanced understanding of single-cell communication through pulsatile cytokine delivery.
Exploitation and Dissemination of Results:
• Technologies: The microfluidic systems developed have significant potential for academic and industrial applications.
• Publications: Findings published in high-impact journals.
• Collaborations: Attracted interest from stakeholders, leading to potential collaborations.
• Conferences and Workshops: Results presented to facilitate knowledge exchange and foster collaborations.
The project met its objectives, leading to innovations and insights into immune cell heterogeneity, with potential impact in vaccine design and cellular therapies for cancer, infectious diseases, and autoimmune disorders.
1. Uncovered multi-layered interferon responses.
2. Enabled automated, high-throughput single-cell screening.
3. Provided precise temporal analysis of cellular signaling.
4. Advanced understanding of single-cell communication through pulsatile cytokine delivery.
Exploitation and Dissemination of Results:
• Technologies: The microfluidic systems developed have significant potential for academic and industrial applications.
• Publications: Findings published in high-impact journals.
• Collaborations: Attracted interest from stakeholders, leading to potential collaborations.
• Conferences and Workshops: Results presented to facilitate knowledge exchange and foster collaborations.
The project met its objectives, leading to innovations and insights into immune cell heterogeneity, with potential impact in vaccine design and cellular therapies for cancer, infectious diseases, and autoimmune disorders.
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.