ILCP Isolation Using CD127+CD117 Markers and Stromal Expansion
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Solution Overview
Problem
Current methods lack effective compositions and methods for isolating and expanding innate lymphoid cell precursors (ILCPs), which are crucial for immune responses but are not well-characterized in humans, particularly for therapeutic applications.
Innovation Solution
Development of compositions comprising purified populations of ILCPs with specific phenotypic markers, such as CD127+CD117+CD3−CRTh2−, and methods for their isolation, expansion, and differentiation into ILC1, ILC2, ILC3, and NK cells using cytokine stimuli and stromal cell-based systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods are used for isolating and expanding innate lymphoid cells, then existing ILC subsets can be obtained, but effective isolation and expansion of ILCPs cannot be achieved
Solution Approach 1:
The patent applies parameter changes by defining specific phenotypic marker combinations (CD127+CD117+CD3−CRTh2−) to identify and isolate ILCPs. This changes the isolation parameters from conventional ILC markers to a specific multi-marker profile that enables precise enrichment of precursor cells with multi-lineage potential, resolving the contradiction between isolation precision and method complexity.
Solution Approach 2:
The patent uses stromal cell-based systems as intermediaries to facilitate ILCP expansion. These stromal cells provide necessary microenvironmental signals and cytokines that enable the expansion of rare ILCPs without requiring complex direct culture conditions, thus simplifying the overall isolation and expansion process while maintaining high precision.
2Reliability
If ILCPs are isolated with high purity using specific phenotypic markers, then multi-potent precursors can be obtained, but the isolation process becomes more complex
Solution Approach 1:
The patent applies segmentation by dividing the isolation process into distinct sequential steps: initial enrichment using CD127 and CD117 markers, followed by negative selection to remove CD3+ T cells and CRTh2+ ILC2s. This segmented approach achieves high purity ILCP populations (≥90%) while managing complexity through modular, stepwise procedures rather than requiring a single complex isolation system.
Solution Approach 2:
The patent employs preliminary action by performing initial enrichment of ILCPs using readily available magnetic beads or flow cytometry targeting CD127+CD117+ cells before applying more complex negative selection steps. This preliminary enrichment reduces the complexity of subsequent purification steps while ensuring high final purity of the ILCP population.
3Productivity
If conventional culture methods are used, then existing ILC subsets can be maintained, but expansion of multi-potent ILCPs is not achieved
Solution Approach 1:
The patent applies universality by developing a culture system using stromal cells that can support the expansion of ILCPs while maintaining their multi-lineage differentiation potential. The stromal cell-based system provides universal support for generating multiple ILC subsets (ILC1, ILC2, ILC3, and NK cells) from a single ILCP population, achieving both high productivity and preserved adaptability simultaneously.
Solution Approach 2:
The patent employs self-service by utilizing stromal cells that naturally provide the necessary cytokines and microenvironmental signals for ILCP expansion and differentiation. The stromal cells autonomously secrete growth factors and create a supportive niche, eliminating the need for complex external supplementation protocols while maintaining the versatility of ILCP differentiation into various ILC lineages.
Data Source
AI summary
Innate lymphoid cells (ILCs) represent innate versions of T helper and cytotoxic T cells that differentiate from committed ILC precursors (ILCP). Still, how ILCP relate to mature tissue-resident ILCs remains unclear. ILCP that are present in the blood and all tested lymphoid and non-lymphoid human tissues were identified. Human ILCP fail to express the signature transcription factors (TF) and cytokine outputs of mature NK cells and ILCs but are epigenetically poised to do so. Human ILCP robustly generate all ILC subsets in vitro and in vivo. While human ILCP express RAR related orphan receptor C (RORC), circulating ILCP can be found in RORC-deficient patients that retain potential for EOMES+ NK cells, T-BET+ ILC1, GATA-3+ ILC2 and for IL-22+ but not for IL-17A+ ILC3. A model of tissue ILC differentiation (‘ILC-poiesis’) is proposed whereby diverse ILC subsets are generated in situ from ILCP in response to environmental stressors, inflammation and infection.