T-cell Separation via CD154 Depletion and Segmentation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods fail to effectively identify and separate regulatory T-cells from conventional T-cells, particularly those specific for antigens, due to contamination issues and the lack of reliable markers, which hinders therapeutic applications for autoimmune and tumor-related immune responses.
Innovation Solution
The use of the CD154 molecule as a marker to deplete CD154+ T-cells from a mixture, combined with additional markers like CD25, GITR, CTLA4, or CD137, allows for the identification and separation of regulatory T-cells, enhancing the purity and yield of activated regulatory T-cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CD25 is used as a marker to identify and separate regulatory T-cells, then regulatory T-cells can be isolated, but conventional T-cells contaminate the isolated population because CD25 is also expressed by activated conventional T-cells
Solution Approach 1:
The patent segments the identification process into two distinct stages: first using CD25 to enrich regulatory T-cells from the total population, then using CD154 to deplete conventional T-cells from the enriched population. This two-step segmentation approach resolves the contradiction by addressing both the need for initial enrichment and the need for final purification, achieving high purity without sacrificing identification specificity.
Solution Approach 2:
The patent extracts the harmful component (conventional T-cells expressing CD154) from the enriched Treg population. By depleting CD154+ cells after CD25 enrichment, the method removes the contamination source while preserving the desired regulatory T-cells, thereby resolving the purity issue without affecting the initial identification accuracy.
2Reliability
If multiple markers are combined to improve separation accuracy, then purity increases, but the complexity of the separation process increases
Solution Approach 1:
The patent employs a dynamic, sequential approach rather than a static simultaneous multi-marker system. The process dynamically progresses through distinct phases: CD25 enrichment first, then CD154 depletion. This dynamic sequencing simplifies the overall complexity compared to attempting simultaneous multi-parameter sorting, while still achieving high purity through the cumulative effect of both markers.
Solution Approach 2:
The patent performs preliminary enrichment using CD25 before the final depletion step with CD154. This preliminary action reduces the burden on the second step by already concentrating the target population, thereby simplifying the overall process complexity while maintaining high purity outcomes.
3Measurement precision
If CD137 is used as an activation marker for Treg, then activated Treg can be identified, but the time window for isolation is limited and contamination with conventional T-cells occurs
Solution Approach 1:
The patent segments the isolation strategy into enrichment (CD25) and depletion (CD154) phases, allowing flexibility in timing. The CD137 marker can be applied at optimal time points within this segmented framework, resolving the time window constraint by decoupling the identification timing from the final isolation step.
Solution Approach 2:
By using CD154 depletion to remove conventional T-cells, the patent extracts the contamination problem separately from the activation marker timing. This allows CD137 to be used for identification at the optimal activation time without being constrained by the need for immediate isolation, as the final purification step can occur after the optimal detection window.
Data Source
AI summary
The present invention relates to a method of identifying and separating non-regulatory T-cells (conventional T-cells) from a mixture comprising regulatory T-cells by using of the CD154 molecule (CD40 ligand) through depletion of CD154+ T-cells from the mixture or in combination with additional positive selection of Treg using markers that are specific for regulatory T-cells, such as for example, CD25, GITR, CTLA4 or markers which are specific for activated regulatory T-cells, such as, for example, CD137, “latent TGF-beta (LAP)”, GARP (LRRC32), CD121a/b, thereby generating a cell composition of activated Treg cells. The invention relates also to a kit comprising an antibody for detecting CD154 and at least one additional antibody for detecting markers for activated or non-activated regulatory T-cells. The antibodies can be coupled to a fluorescent dye or magnetic microparticles.