Sortase-Mediated Intercellular Labeling for Immune Cell Tracking
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Solution Overview
Problem
Current methods lack an effective way to track interactions between immune cells, such as B cells and follicular T helper cells, in vivo, which is crucial for understanding affinity maturation and antibody selection, due to the inability to determine the extent of cell interactions in a living animal.
Innovation Solution
An intercellular labeling method using sortase-mediated labeling, where cells expressing specific polypeptides interact via receptor-ligand pairs, allowing for the detection of interactions using a labeled peptide with a detectable label, enabling tracking of cell interactions in vitro, ex vivo, and in vivo.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods are used to study immune cell interactions, then basic immune response understanding is maintained, but the ability to track and measure cell interactions in vivo is lost
Solution Approach 1:
The patent introduces sortase enzyme as an intermediary that mediates the labeling process. The sortase enzyme is expressed on one cell type (e.g., T cells) and recognizes a specific peptide sequence on interacting cells (e.g., B cells), enabling indirect detection of cell-cell interactions through enzymatic labeling with detectable substrates.
Solution Approach 2:
The patent changes the chemical parameter of cell surface polypeptides by incorporating sortase recognition sequences (specific amino acid sequences) that can be covalently modified by the sortase enzyme. This parameter change enables selective labeling of interacting cells without affecting other cells, achieving precise measurement capability.
2Loss of information
If sortase-mediated labeling is implemented, then cell interaction detection capability is improved, but the complexity of the labeling system increases
Solution Approach 1:
The sortase enzyme system is designed to be universally applicable to multiple cell types and interaction pairs. The same sortase enzyme can label different target cells expressing the recognition sequence, and the system can be adapted to study various immune cell interactions (T-B cell, dendritic cell-T cell, etc.) using the same fundamental mechanism.
Solution Approach 2:
The patent uses detectable substrate analogs that mimic the natural sortase recognition sequence but include detectable labels (fluorophores, biotin, etc.). These copied molecules allow indirect detection of the enzymatic activity and thus the cell interactions without directly labeling the cells themselves.
3Reliability
If in vivo tracking of cell interactions is achieved, then understanding of affinity maturation is improved, but the technical requirements for the system increase
Solution Approach 1:
The sortase recognition sequences are pre-incorporated into the cell surface polypeptides before the interactions occur. This preliminary preparation ensures that when cell-cell contact happens, the labeling can occur immediately without requiring additional preparation steps, enhancing the reliability of interaction tracking in vivo.
Solution Approach 2:
The system enables self-labeling of interacting cells through the natural cell-cell interaction process itself. When cells interact via their native receptors and ligands, the sortase enzyme on one cell automatically labels the other cell through the recognition sequence, making the labeling process self-driven by the biological interaction rather than requiring external intervention.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method allows for the visualization and measurement of immune cell interactions, facilitating the identification of high-affinity B cell receptors and understanding the selection processes in germinal centers, and can be applied to various biological studies and disease analysis.
Implementation Method 1
the sortase, or the active fragment thereof, links the peptide to the first polypeptide, thereby labeling the first cell expressing the first polypeptide
Data Source
AI summary
An sortase-mediated intercellular labeling method allowing for tracking ligand-receptor interaction both in vitro and in vivo; and uses thereof for tracking molecule interactions both in vitro and in vivo, identifying modulators of ligand-receptor interaction, identifying potential binding partners of a protein of interest, identifying B cells expressing high affinity B cell receptors to antigens, and identifying the antigen to which a T cell of interest binds.