Foxp3+ Treg Isolation via IL-34 Capture Moiety

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Solution Overview

Problem

Current methods for isolating Foxp3+ regulatory T cells from biological samples are contaminated with effector T cells due to the lack of specific cell surface markers, particularly for CD8+ Foxp3+ Tregs, which are crucial for maintaining tolerance and immune suppression.

Innovation Solution

A method involving the use of a capture moiety that binds to cell surface molecules and Interleukin-34 (IL34), allowing for the labeling and isolation of Foxp3+ Treg cells by capturing IL34-expressing lymphocytes, thereby achieving high purity and specificity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of manufacture

If cell surface markers such as CD25 are used for isolating Foxp3+ Treg cells, then the isolation process is simple and markers are readily available, but the isolated population is significantly contaminated with effector T cells (up to half of the population)

Engineering Contradiction:

Improveease of isolationVSAvoidpurity of isolated Treg cells

Core Design Contradiction:

Ease of manufactureVSManufacturing precision

Solution Approach 1:

The isolation method segments the Treg cell population into distinct subsets based on IL-34 expression levels. By using flow cytometry to detect and sort cells with high IL-34 expression, the method separates true Foxp3+ Treg cells from contaminating effector T cells, achieving both ease of isolation and high purity simultaneously

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention introduces IL-34 as an intermediary marker to indirectly identify Foxp3+ Treg cells. Since IL-34 is specifically produced by Foxp3+ Treg cells and not by effector T cells, it serves as a reliable intermediary indicator that enables precise isolation without requiring direct detection of Foxp3 intracellular protein

Inventive Principle:

Principle #24Intermediary (Mediator)

2Adaptability or versatility

If conventional markers like CD4 and CD25 are employed for Treg isolation, then the methodology is well-established and easy to implement, but the specificity is insufficient as these markers are not restricted to Tregs

Engineering Contradiction:

Improvemethodological establishednessVSAvoidspecificity of Treg identification

Core Design Contradiction:

Adaptability or versatilityVSMeasurement precision

Solution Approach 1:

The invention changes the detection parameter from conventional cell surface markers (CD4, CD25) to an intracellular cytokine marker (IL-34). This parameter change increases specificity because IL-34 expression is uniquely associated with Foxp3+ Treg cells, while maintaining adaptability through the use of standard flow cytometry techniques that can detect intracellular cytokines

Inventive Principle:

Principle #35Parameter changes

3Ease of manufacture

If Foxp3+ Treg cells are isolated using current methods, then the process can be performed with available reagents, but the resulting population contains significant effector T cell contamination that compromises therapeutic efficacy

Engineering Contradiction:

Improveavailability of reagentsVSAvoidtherapeutic effectiveness

Core Design Contradiction:

Ease of manufactureVSReliability

Solution Approach 1:

The method performs preliminary enrichment of Foxp3+ Treg cells by selecting for high IL-34 expression before final isolation. This preliminary action removes contaminating effector T cells that do not express IL-34, ensuring that the isolated population has the required therapeutic effectiveness while still using readily available reagents and flow cytometry technology

Inventive Principle:

Principle #10Preliminary action

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 yields a population of Foxp3+ Treg cells that is virtually free from contaminating effector T cells, facilitating their expansion and use in inducing antigen-specific immune tolerance, particularly in autoimmunity, allergy, and transplantation contexts.

Implementation Method 1

coupling the surface of PBMC or lymphocytes to a capture moiety which binds to the cell through a cell surface molecule and to Interleukin-34 (IL34)

Methodology Applied

Scientific EffectSpecific binding: Adsorption

Data Source

PatentUS10591465B2Methods and kits for labeling, detection and isolation of Foxp3<sup>+</sup> regulatory T cells, isolated population of Foxp3<sup>+</sup> regulatory T cells thus obtained and uses thereof

Publication Date: 2020.03.17 UNIV DE NANTES

AI summary

Disclosed is a method for labeling, detecting and/or isolating Foxp3+ Treg cells from a biological sample containing peripheral blood mononuclear cells (PBMC) or lymphocytes including the following steps of: (i) coupling the surface of PBMC or lymphocytes to a capture moiety which binds to the cell through a cell surface molecule and to interleukin-34 (IL34), (ii) culturing the lymphocytes under conditions wherein IL34 is secreted, released and specifically captured by the capture moiety, (iii) labeling the IL34 expressing lymphocytes with a label moiety, and (iv) optionally detecting and/or isolating the IL34 expressing lymphocytes which are Foxp3+ Treg cells. Also disclosed is an isolated population of Foxp3+ Treg cells obtainable by the method and uses thereof.