Single-Cell Topographical Analysis Reveals Biomechanical Characteristics of Cytotoxic T Cells

Introduction

In recent years, research on how the immune system functions in different mechanochemical environments has shown that immune cells dynamically alter their shape and exert forces on their surroundings to sense physical parameters and activate immune responses. These physical parameters significantly affect cell gene expression, metabolism, and meso-scale cell behavior. Specifically, cytotoxic T cells (CTLs) kill infected or transformed target cells by releasing perforin and granzymes, and this secretory behavior is closely related to mechanical action. However, how the forces derived from CTLs precisely locate the release of perforin and granzymes and how these forces affect the target cell membrane remain unresolved questions. To address this, the authors used super-resolution traction force microscopy (TFM) to compare immune synapses formed by CTLs with those formed by other T cell subsets and macrophages, aiming to reveal unique force output patterns and understand the functions of CTLs in potential mechanical environments.

Source of the Paper

This paper was completed by Miguel de Jesus et al., with authors from Memorial Sloan Kettering Cancer Center, Wageningen University & Research, University of Washington, among other research institutions. The paper was published in Science Immunology on June 28, 2024, titled “Single-cell topographical profiling of the immune synapse reveals a biomechanical signature of cytotoxicity.”

Research Process

Experimental Methods

The study utilized a three-dimensional traction force microscopy system (3D TFM) to analyze the mechanical characteristics of interactions by inducing T cells to form synapses with deformable polyacrylamide particles (DAAM particles, diameter 13 μm, stiffness 300 Pa). DAAM particles were functionalized to induce the formation of T cell immune synapses and imaged using high-speed structured illumination microscopy (SIM). Triangulation was used to reconstruct the surface deformation of each particle, making the physical deformation induced by CTL synapses visible.

Data Analysis and Algorithms

During the analysis, the authors used a series of custom MATLAB scripts to achieve the three-dimensional shape reconstruction of the particles and performed topographical analysis using Zernike polynomials to represent each immune synapse as a series of spatial frequency spectra. This method allowed the arrangement of topologies according to similarities, revealing unique mechanical characteristics of cytotoxic T cells compared to other immune cells.

Results

Analysis of Results

The study found that CTLs not only spread on DAAM particles but also pressed into them, forming a synapse “crater” about 10μm in diameter in the contact area. This “crater” includes a positively curved peripheral edge (rim area) and a central concave region (crater floor) with local elevations and depressions. Quantitative analysis of the deviations of the DAAM particle shape from an ideal sphere confirmed that CTLs could induce significant particle compression within 5 minutes of contact and maintain it for over 30 minutes. Analysis of the f-actin (F-actin) distribution showed that these topographical features were caused by local cytoskeletal restructuring, and the actin polymerization inhibitor Latrunculin A almost completely eliminated the target compression.

To assess the relationship between these structures and cytotoxic release, the study labeled Lamp1 to observe granule dynamics in real time, finding that fusion and disappearance of granules primarily occurred on the crater floor, indicating that the release of perforin and granzymes was concentrated in this region. A computational model based on continuum elasticity theory simulated the F-actin driven synapse forces, suggesting that the topography of the synapse can maximize the efficiency of cytotoxicity.

Additionally, for F-actin protrusions, it was found that clustered structures were more effective in deforming the target surface and providing more membrane area for toxic granule fusion and release than dispersed small protrusions. This combined strategy may explain the specific mechanical output patterns formed by CTLs during evolution.

Conclusions and Significance

This study reveals the unique biomechanical characteristics of immune synapses formed by CTLs, providing a new perspective on understanding CTL killing mechanisms. Specifically, the efficiency of CTL killing relies not only on chemical secretions but also on complex mechanical actions exerted on the target cell. Further experiments showed that this mechanical input pattern is optimized by adapting to target physical properties. These findings indicate that the mechanical interaction forms between immune cells reflect their specific functions, and interface mechanical patterns can be used to distinguish different immune cell subsets.

Highlights and Innovations

The highlights of the study are: 1. Close link between mechanics and function: Revealed a strong correlation between cytotoxicity and specific mechanical output patterns (compressive forces and local protrusions). 2. High-resolution imaging technology: Utilized super-resolution 3D TFM technology for the first time to clearly present the mechanical interactions between CTLs and target particles. 3. Combination of computational models and experimental validation: By integrating computational models with experimental data, the study elucidated the relationship between synapse mechanical output and cytotoxicity efficiency and identified the key role of F-actin patterns in synapse mechanical properties. 4. Introduction of topographical complexity analysis: Introduced Zernike polynomials and their rotational invariance to quantify the topographical complexity of immune synapses and compare differences between cells.

This study not only provides essential foundational knowledge in biomechanics and cytotoxicity but also aids in developing intervention strategies targeting immune cell function, holding significant applications for immunotherapy and pathological research.