Application of Mass Cytometry Platforms to Solid Organ Transplantation

Abstract

Transplantation serves as the cornerstone of treatment for patients with end-stage organ disease. The prevalence of complications, such as allograft rejection, infection, and malignancies, underscores the need to dissect the complex interactions of the immune system at the single-cell level. In this review, we discuss studies using mass cytometry or cytometry by time-of-flight, a cutting-edge technology enabling the characterization of immune populations and cell-to-cell interactions in granular detail. We review the application of mass cytometry in human and experimental animal studies in the context of transplantation, uncovering invaluable contributions of the tool to understanding rejection and other transplant-related complications. We discuss recent innovations that have the potential to streamline and standardize mass cytometry workflows for application to multisite clinical trials. Additionally, we introduce imaging mass cytometry, a technique that couples the power of mass cytometry with spatial context, thereby mapping cellular interactions within tissue microenvironments. The synergistic integration of mass cytometry and imaging mass cytometry data with other omics data sets and high-dimensional data platforms to further define immune dynamics is discussed. In conclusion, mass cytometry technologies, when integrated with other tools and data, shed light on the intricate landscape of the immune response in transplantation. This approach holds significant potential for enhancing patient outcomes by advancing our understanding and facilitating the development of new diagnostics and therapeutics.

Figure 1:. A. Schematic diagram of mass cytometry workflow:

Single-cell suspensions are combined with heavy metal-labeled antibodies that target specific markers. To detect intracellular proteins standard fixation and permeabilization protocols are utilized. After undergoing several washing steps, the antibody-labeled cells are introduced into the mass cytometer. In this stage, the suspension is transformed into small droplets and then introduced into an inductively coupled plasma (ICP), which disintegrates the droplets’ contents into a cloud of elemental ions. Ions with lower atomic masses are eliminated through a mass filter called a quadrupole, while the remaining ions which are bound to the corresponding probes are measured using an orthogonal time of flight (TOF) mass spectrometric detection system. The obtained ion counts are used to determine the quantities of bound antibodies, thereby providing information on the single-cell abundance. B. Schematic diagram of mass cytometry imaging workflows: To analyze tissue sections, either formalin -fixed paraffin-embedded (FFPE) or cryopreserved sections are stained using heavy metal-labeled antibodies. In MIBI, the stained sections are placed in the MIBI analyzer and placed in a vacuum chamber. The MIBI instrument then scans the tissue sections with a primary ion beam. As the primary ion beam collides with the stained section, secondary ions are liberated from the sample, including those introduced through the binding of the heavy metal-labeled antibodies. This cloud of secondary ions is focused through lenses and introduced into an orthogonal TOF mass spectrometer. In IMC, the stained sections are placed into the IMC analyzer, where a laser systematically ablates the tissue pixel by pixel. The released particles are then introduced into an analyzer, where they are ionized in an ICP and quantified using an orthogonal TOF mass spectrometer. In both techniques, the detected signal is integrated and translated into ion counts per pixel, resulting in multidimensional images that can be directly visualized or further analyzed using various image analysis pipelines.

Figure 1:. A. Schematic diagram of mass cytometry workflow:

Single-cell suspensions are combined with heavy metal-labeled antibodies that target specific markers. To detect intracellular proteins standard fixation and permeabilization protocols are utilized. After undergoing several washing steps, the antibody-labeled cells are introduced into the mass cytometer. In this stage, the suspension is transformed into small droplets and then introduced into an inductively coupled plasma (ICP), which disintegrates the droplets’ contents into a cloud of elemental ions. Ions with lower atomic masses are eliminated through a mass filter called a quadrupole, while the remaining ions which are bound to the corresponding probes are measured using an orthogonal time of flight (TOF) mass spectrometric detection system. The obtained ion counts are used to determine the quantities of bound antibodies, thereby providing information on the single-cell abundance. B. Schematic diagram of mass cytometry imaging workflows: To analyze tissue sections, either formalin -fixed paraffin-embedded (FFPE) or cryopreserved sections are stained using heavy metal-labeled antibodies. In MIBI, the stained sections are placed in the MIBI analyzer and placed in a vacuum chamber. The MIBI instrument then scans the tissue sections with a primary ion beam. As the primary ion beam collides with the stained section, secondary ions are liberated from the sample, including those introduced through the binding of the heavy metal-labeled antibodies. This cloud of secondary ions is focused through lenses and introduced into an orthogonal TOF mass spectrometer. In IMC, the stained sections are placed into the IMC analyzer, where a laser systematically ablates the tissue pixel by pixel. The released particles are then introduced into an analyzer, where they are ionized in an ICP and quantified using an orthogonal TOF mass spectrometer. In both techniques, the detected signal is integrated and translated into ion counts per pixel, resulting in multidimensional images that can be directly visualized or further analyzed using various image analysis pipelines.

Figure 2:. Schematic diagram of Maxpar Direct Immune Profiling Assay workflow:

Whole blood or PBMC are added to MDIPA tubes containing a 30‐marker panel of lyophilized antibodies for phenotyping immune cells. If samples are to be frozen and transported, the SMART TUBE stabilizer is added to the MDIPA tubes prior to freezing. Samples are fixed after thawing and treated with an intercalator. Stained samples are then analyzed on a CyTOF instrument. The immune cell populations are identified and quantitated using the Pathsetter^™ software. (Image adapted from standardbio.com)