Taking Cytometry to Another Dimension
Authors:   , Verhoeff Jan 
Published: February 2025

This thesis explores the application of high-dimensional cytometry in immunology research. Modern cytometers measure over twenty phenotypic parameters on individual cells and analyze thousands to millions of cells per sample. This high throughput allows for the identification of rare cell populations and enumeration of various cell types, crucial in immunology due to the diversity of immune cells. These cells perform functions based on the expression and modification of proteins, responding to stimuli like pathogens by altering cell phenotypes and activating or inhibiting certain processes.

Cytometry relies on reporters attached to labeling molecules that bind targets on or in cells. Targets are selected based on their association with specific phenotypes or cellular functions. Quantifying the reporters reflects the amount of target proteins present, allowing for phenotyping based on marker sets. However, environmental factors and experimental variables can cause variability, so standardization and controls are essential for reproducibility.

This thesis focuses on advancing experimental design, execution, and data analysis in cytometry. The first section addresses the experimental cycle, while the second applies high-dimensional cytometry to immunological studies. Chapter 1 provides an overview of the immune system, cytometry technologies, and experimental designs to improve consistency.

Chapter 2 details the development and validation of HyperMAP (Hyperloaded MetAlcomplexed Polymers) molecules, which allow precise control over ion incorporation and enable the use of 21 elements. These molecules were attached to antibodies, which retained their binding capabilities to immune cells, expanding the potential for spectral mass cytometry. This method enables the analysis of larger panels and improves signal deconvolution for multi-isotopic metals.

In Chapter 3, we describe the design and optimization of a mass cytometry panel for phenotyping immune cells from the brain, spleen, and bone marrow of mice. The panel consisted of 34 antibodies, optimized for signal and concentration. The results from these experiments are shared to help researchers skip optimization steps.

Chapter 4 introduces a computational method for evaluating cytometry clustering results. While clustering methods are expanding, there is a lack of metrics to assess their effectiveness. We use Hartigan’s dip test to detect multiple distributions within a peak, allowing for better cluster validation. The method identifies the optimal number of clusters, improving data interpretation.

Chapter 5 explores how altered sugar structures (glycosylation) on tumor cells affect the immune system. We observed increased levels of immature O-linked glycans on glioblastoma cells, which interact with the MGL receptor on CD163+ macrophages in the brain, contributing to immunosuppression. This study suggests that glycan alterations in glioblastoma tumors create an immunosuppressive environment.

In Chapter 6, we studied the immune composition of peripheral blood in 169 patients with intracranial tumors, including glioblastoma and meningiomas. Patients treated with dexamethasone, an immunosuppressive drug, exhibited altered immune responses, with a higher percentage of memory B cells and fewer alternative monocytes. These findings emphasize the importance of immunological screening before immunotherapy in glioblastoma treatment.

Chapter 7 investigates immune changes in peripheral blood mononuclear cells (PBMCs) of 40 community-acquired pneumonia (CAP) patients, 31 controls, and 35 COVID-19 patients. Spectral flow cytometry revealed 86 distinct metaclusters, with 19 being differentially abundant between CAP patients and controls. Changes in classical monocytes were associated with immunosuppression, and the presence of specific monocyte subtypes predicted longer recovery times. The immune response to CAP was similar to COVID-19.

Chapter 8 reviews the central theme of this thesis—high-dimensional cytometry. The experimental cycle is discussed, with recommendations for proper execution. The development of cytometry as a technique is contextualized, highlighting its role in immunology research and its potential for future applications in systems immunology, particularly in the emerging field of cytOMICs.