Project description:There is a critical unmet need to image 2D materials within single cells and tissues. Here we propose a versatile label-free single-cell detection strategy based on mass cytometry. We use single-cell mass cytometry by time-of-flight (CyTOF), imaging mass cytometry, and multiplexed ion beam imaging by time-of-flight (MIBI-TOF) with three representative MXenes. Nb4C3, Mo2Ti2C3, and Ta4C3 were selected to ensure mass-detection within the range and to avoid overlap with currently available tags. We demonstrated their biocompatibility, detection, and quantification in 16 primary human immune cell subpopulations also showing the impact of MXene lateral size on the detection signal and cell binding. We report the biological and immune functional compatibility of MXenes. In vivo biodistribution experiments in mice using a mixture of MXenes revealed their presence in liver, blood, spleen, and lungs. Lastly, we applied MIBI-TOF to capture the presence of MXenes in different organs. The label-free detection of 2D materials by mass cytometry at the single cell level, on multiple subpopulations and in multiple organs in vivo opens exciting new opportunities in biomedicine.
Project description:Macrophages and dendritic cells are phagocytes present in almost all tissues in mammals and play a pivotal role for tissue homeostasis and during immune responses. Liver phagocytes play a pivotal role in host immune responses and exquisite mechanisms are necessary to govern the density and the location of the different hepatic leukocytes. However in catastrophic events including trauma, infections or toxin ingestion, many of the liver phagocytes can be wiped out leaving large areas devoid of these cells. Here we used a unique combination of mass cytometry (CyTOF), gene expression and liver intravital approaches to precisely determine phagocytic populations within the liver and the functional consequences of their replenishment by myeloid precursors. While Kupffer cells were exclusively located in the sinusoidal lumen, we identified a population of dendritic cells that was mainly located under the liver capsule. After full depletion of dendritic and Kupffer cells, intravascular myeloid precursors replenished location, density and function of both populations. However, these emergency repopulated livers were dysfunctional in their ability to respond to injury and to clear bacteria for at least 30 days. After this âeducational periodâ, new phagocytes returned to normal response to injury and bacterial trapping. Conclusions: Our data shed light on the liverâs ability to locally shape phagocyte precursors into two vastly different immune cells localized to two fundamentally different tissue compartments. 4 different cell types were isolated from mice, RNA was extracted, and gene expression was measured using the Nanostring nCounter Mouse Immunology Panel
Project description:<p>A more complete and holistic view on host–microbe interactions is needed to understand the physiological and cellular barriers that affect the efficacy of drug treatments and allow the discovery and development of new therapeutics. Here, we developed a multimodal imaging approach combining histopathology with mass spectrometry imaging (MSI) and same section imaging mass cytometry (IMC) to study the effects of <em>Salmonella Typhimurium</em> infection in the liver of a mouse model using the <em>S. Typhimurium</em> strains SL3261 and SL1344. This approach enables correlation of tissue morphology and specific cell phenotypes with molecular images of tissue metabolism. IMC revealed a marked increase in immune cell markers and localization in immune aggregates in infected tissues. A correlative computational method (network analysis) was deployed to find metabolic features associated with infection and revealed metabolic clusters of acetyl carnitines, as well as phosphatidylcholine and phosphatidylethanolamine plasmalogen species, which could be associated with pro-inflammatory immune cell types. By developing an IMC marker for the detection of <em>Salmonella</em> LPS, we were further able to identify and characterize those cell types which contained <em>S. Typhimurium</em>.</p>
Project description:Macrophages and dendritic cells are phagocytes present in almost all tissues in mammals and play a pivotal role for tissue homeostasis and during immune responses. Liver phagocytes play a pivotal role in host immune responses and exquisite mechanisms are necessary to govern the density and the location of the different hepatic leukocytes. However in catastrophic events including trauma, infections or toxin ingestion, many of the liver phagocytes can be wiped out leaving large areas devoid of these cells. Here we used a unique combination of mass cytometry (CyTOF), gene expression and liver intravital approaches to precisely determine phagocytic populations within the liver and the functional consequences of their replenishment by myeloid precursors. While Kupffer cells were exclusively located in the sinusoidal lumen, we identified a population of dendritic cells that was mainly located under the liver capsule. After full depletion of dendritic and Kupffer cells, intravascular myeloid precursors replenished location, density and function of both populations. However, these emergency repopulated livers were dysfunctional in their ability to respond to injury and to clear bacteria for at least 30 days. After this “educational period”, new phagocytes returned to normal response to injury and bacterial trapping. Conclusions: Our data shed light on the liver’s ability to locally shape phagocyte precursors into two vastly different immune cells localized to two fundamentally different tissue compartments.
Project description:Kilian2024 - Immune cell dynamics in Cue-Induced Extended Human Colitis Model
Single-cell technologies such as scRNA-seq and flow cytometry provide critical insights into immune cell behavior in inflammatory bowel disease (IBD). However, integrating these datasets into computational models for dynamic analysis remains challenging. Here, Kilian et al., (2024) developed a deterministic ODE-based model that incorporates these technologies to study immune cell population changes in murine colitis. The model parameters were optimized to fit experimental data, ensuring an accurate representation of immune cell behavior over time. It was then validated by comparing simulations with experimental data using Pearson’s correlation and further tested on independent datasets to confirm its robustness. Additionally, the model was applied to clinical bulk RNA-seq data from human IBD patients, providing valuable insights into immune system dynamics and potential therapeutic strategies.
Figure 4c, obtained from the simulation of human colitis model is highlighted here.
This model is described in the article:
Kilian, C., Ulrich, H., Zouboulis, V.A. et al. Longitudinal single-cell data informs deterministic modelling of inflammatory bowel disease. npj Syst Biol Appl 10, 69 (2024). https://doi.org/10.1038/s41540-024-00395-9
Abstract:
Single-cell-based methods such as flow cytometry or single-cell mRNA sequencing (scRNA-seq) allow deep molecular and cellular profiling of immunological processes. Despite their high throughput, however, these measurements represent only a snapshot in time. Here, we explore how longitudinal single-cell-based datasets can be used for deterministic ordinary differential equation (ODE)-based modelling to mechanistically describe immune dynamics. We derived longitudinal changes in cell numbers of colonic cell types during inflammatory bowel disease (IBD) from flow cytometry and scRNA-seq data of murine colitis using ODE-based models. Our mathematical model generalised well across different protocols and experimental techniques, and we hypothesised that the estimated model parameters reflect biological processes. We validated this prediction of cellular turnover rates with KI-67 staining and with gene expression information from the scRNA-seq data not used for model fitting. Finally, we tested the translational relevance of the mathematical model by deconvolution of longitudinal bulk mRNA-sequencing data from a cohort of human IBD patients treated with olamkicept. We found that neutrophil depletion may contribute to IBD patients entering remission. The predictive power of IBD deterministic modelling highlights its potential to advance our understanding of immune dynamics in health and disease.
This model was curated during the Hackathon hosted by BioMed X GmbH in 2024.
Project description:Dr. Christopher Contag lab's aim is to, investigate the molecular interactions that underlie CIK cell homing from the bloodstream to tumor tissue using a combination of gene expression profiling, flow cytometry, and in vivo molecular imaging. Collectively, these proposed experiments will define the repertoire of molecules employed in CIK cell recognition of the tumor vasculature and guide the development of improved CIK cell therapies for use in cancer patients. A promising new direction in cancer therapy is the use of cytokine-induced killer (CIK) cells as broadly active tumoricidal agents. CIK cells derive from blood samples stimulated ex vivo, and these activated immune cells are capable of recognizing and destroying a plethora of tumor targets in vivo. In fact, CIK cells have demonstrated efficacy in clinical trials to treat patients with hepatoma, renal cell cancer, and hematological malignancies. Despite the remarkable clinical promise of this therapy, little is known about the mechanisms that govern CIK cell migration to tumor tissue in vivo. Understanding the trafficking patterns of these potent immune cells is of critical importance to advancing their use in humans. I aim to investigate the molecular interactions that underlie CIK cell homing from the bloodstream to tumor tissue using a combination of gene expression profiling, flow cytometry, and in vivo molecular imaging. Collectively, these proposed experiments will define the repertoire of molecules employed in CIK cell recognition of the tumor vasculature and guide the development of improved CIK cell therapies for use in cancer patients. RNA from three groups (1,2,3) were sent to Microarray Core E. Group one samples: Splenocytes were obtained from BALB/c mice, and the T cells were isolated using a negative isolation kit (magnetic bead separation). For group 2 and 3 samples: The isolated splenocytes were treated with IFN-gamma overnight. The cells were then added to anti-CD3 coated flasks and stimulated with IL-2. Fresh IL-2 was added to the media every 3 days, and the cells were harvested 12 days post-isolation. The RNA was labeled and hybridized to Glyco_v3 arrays.
Project description:Immune profiles were performed retrospectively in highly sensitized kidney transplant candidates Our hypothesis was that baseline differences in immune profiles could help identify candidates that respond to desensitization therapy. Single-cell mass cytometry by time-of-flight (CyTOF) phenotyping, gene arrays, and phosphoepitope flow cytometry were performed in 20 highly sensitized kidney transplant candidates undergoing desensitization therapy.
Project description:Regulated chromatin states control genome accessibility and thus influence gene expression. Here we report an analysis pipeline termed ATAC-mass that capitalizes on isotopic labeling to detect the accessible genome by multiplexed ion beam imaging (MIBI) and mass cytometry. With MIBI the accessible genome can be visualized at approximately 100-nm resolution simultaneously with metabolic labeling to enable multi-parameter three-dimensional imaging of nuclear features. Extension of this approach to non-spatial mass cytometry enabled the simultaneous measurement of multiple parameters and total genome accessibility in millions of individual cells. We used ATAC-mass to analyze natural killer cells after stimulation with interleukin (IL)-12 or IL-18 -- demonstrating that IL-18 treatment leads to increased total genome accessibility. Analysis of the spatial organization of open chromatin suggest that IL-12 and IL-18 both induce an increase in chromatin accessibility in noncompacted DNA regions. Deep sequencing of the genomic distribution of open chromatin revealed that IL-18 increased the accessibility of quiescent enhancers whereas genomic loci that become more accessible by IL-12 stimulation are mainly localized in the active promoter regions. This integration of epigenomics, proteomics and high-resolution imaging at the single-cell level provides a tool that can enhance our appreciation of the molecular mechanisms underlying gene regulation.
Project description:Dr. Christopher Contag lab's aim is to, investigate the molecular interactions that underlie CIK cell homing from the bloodstream to tumor tissue using a combination of gene expression profiling, flow cytometry, and in vivo molecular imaging. Collectively, these proposed experiments will define the repertoire of molecules employed in CIK cell recognition of the tumor vasculature and guide the development of improved CIK cell therapies for use in cancer patients. A promising new direction in cancer therapy is the use of cytokine-induced killer (CIK) cells as broadly active tumoricidal agents. CIK cells derive from blood samples stimulated ex vivo, and these activated immune cells are capable of recognizing and destroying a plethora of tumor targets in vivo. In fact, CIK cells have demonstrated efficacy in clinical trials to treat patients with hepatoma, renal cell cancer, and hematological malignancies. Despite the remarkable clinical promise of this therapy, little is known about the mechanisms that govern CIK cell migration to tumor tissue in vivo. Understanding the trafficking patterns of these potent immune cells is of critical importance to advancing their use in humans. I aim to investigate the molecular interactions that underlie CIK cell homing from the bloodstream to tumor tissue using a combination of gene expression profiling, flow cytometry, and in vivo molecular imaging. Collectively, these proposed experiments will define the repertoire of molecules employed in CIK cell recognition of the tumor vasculature and guide the development of improved CIK cell therapies for use in cancer patients.