Antigen-Specific Treg Manufacturing via Fluorescent Dye Dilution Sorting
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Solution Overview
Problem
Current methods for producing T regulatory (Treg) lymphocytes for clinical use are limited by their polyspecificity, which reduces effectiveness and increases systemic side effects, as they target multiple antigens rather than specific ones, and there is a need for a safe and efficient protocol to expand antigen-specific Treg cells while maintaining their suppressor activity.
Innovation Solution
A process involving autologous monocytes loaded with antigens, T lymphocytes stained with fluorescent dyes, and incubation with anti-CD154 and anti-CD28 antibodies to generate antigen-specific T lymphocytes, which are then sorted based on low intracellular dye intensity to enhance specificity and suppressive function.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If Treg lymphocytes are expanded using conventional polyclonal methods, then sufficient cell numbers are obtained for clinical use, but the cells exhibit polyspecificity that reduces effectiveness and increases systemic side effects
Solution Approach 1:
The patent segments the polyclonal Treg population into antigen-specific subsets by sorting cells based on their proliferation response to specific antigens. This is achieved by co-culturing Tregs with antigen-presenting cells loaded with specific antigens, then sorting proliferating antigen-specific Tregs using flow cytometry based on dilution of intracellular fluorescent dyes.
Solution Approach 2:
The patent applies preliminary action by pre-loading antigen-presenting cells with specific antigens before co-culture with Treg lymphocytes. This allows selective activation and proliferation of antigen-specific Tregs in advance of the sorting step, enabling enrichment of the desired cell population.
2Adaptability or versatility
If polyclonal Treg lymphocytes are used for therapy, then broad immunosuppressive coverage is achieved, but systemic side effects increase due to non-specific suppression
Solution Approach 1:
The patent applies local quality by making the immunosuppressive effect specific to particular antigens rather than universal. Antigen-specific Tregs are generated to target specific self-antigens involved in autoimmune diseases or specific alloantigens in transplantation, providing localized immunosuppression at the molecular level while sparing other immune responses.
Solution Approach 2:
The patent extracts the harmful polyspecificity from the Treg population by sorting out only those cells that specifically recognize and respond to the target antigen. This leaves behind non-specific suppressive effects while retaining the desired antigen-specific regulatory function.
3Manufacturing precision
If Treg lymphocytes are sorted based on fluorescence intensity, then antigen-specific cells are enriched, but the sorting process complexity increases
Solution Approach 1:
The patent uses intracellular fluorescent dyes (such as CFSE or Violet) as intermediaries to mark Treg cells before activation. These dyes dilute with each cell division, providing a simple readout mechanism where proliferating antigen-specific Tregs show reduced fluorescence intensity. This intermediary marker system simplifies the sorting process compared to direct antigen detection methods.
Solution Approach 2:
The patent replaces complex mechanical or biochemical sorting methods with fluorescence-based sorting. Instead of using complex devices to physically separate cells based on antigen recognition, the method uses optical detection of fluorescent dye dilution, which can be easily measured by flow cytometry and sorted using standard fluorescence-activated cell sorting equipment.
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 produces antigen-specific Treg lymphocytes with preserved regulatory phenotype and enhanced suppressive activity, effectively targeting specific antigens and reducing systemic side effects, thereby improving treatment efficacy for autoimmune diseases and allergic reactions.
Implementation Method 1
T regulatory or T effector lymphocytes to be generated are suspended in PB S and stained intracellularly with a fluorescent dye
Implementation Method 2
the co-culture of T regulatory or T effector lymphocytes with CD14+ monocytes is coincubated with anti-CD154 and anti-CD28 antibodies
Implementation Method 3
the lymphocytes are suspended in the culture medium with gamma-irradiated autologous CD14+ monocytes loaded with antigen
Data Source
AI summary
The invention relates to a new process for manufacturing in vitro antigen-specific T lymphocytes (CellTrAg), marked with intracellular dye and expanded in the presence of monocytes loaded with the antigen and subsequently sorted based on the low intensity of intracellular dye where the low intensity of fluorescence is a marker of antigen-specificity in that a loss of fluorescence correlated with the intensity of proliferation. The antigen specificity is assessed in functional tests in which antigen-specific lymphocytes sorted on the basis of low fluorescence are more active than non-specific lymphocytes with high fluorescence during sort; activity is defined in the case of regulatory T lymphocytes as inhibition of effector lymphocyte function and in the case of T effector lymphocytes as enhancing the characteristics of these cells such as proliferation, production of cytokines and cytotoxic factors.