Initial or continuous coculture with umbilical cord-derived mesenchymal stromal cells facilitates in vitro expansion of human regulatory T-cell subpopulations

Area of Science:

• Immunology
• Cell Therapy
• Regenerative Medicine

Background:

• Ex vivo expanded regulatory T cells (Tregs) show promise for treating immune-mediated diseases.
• Current Treg expansion methods face challenges in achieving consistent yield and purity.
• Human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) are being explored to improve cell manufacturing.

Purpose of the Study:

• To evaluate the enhancement of primary human Treg and Treg subpopulation expansion using hUC-MSC coculture.
• To assess the impact of hUC-MSC coculture on Treg suppressive potency and marker expression.
• To investigate the differential effects of hUC-MSC coculture on naive and non-naive Treg subpopulations.

Main Methods:

• Primary human total Tregs and FACS-purified Treg subpopulations were cultured with or without hUC-MSCs.
• Cultures were supplemented with anti-CD3/anti-CD28, interleukin-2, and rapamycin for 14-21 days.
• Treg expansion, T effector cell (Teff) proliferation suppression, and marker expression (HLA-DR, CD39, ICOS, Foxp3, Helios) were analyzed using FACS.

Main Results:

• hUC-MSC coculture resulted in a 4-fold greater expansion of total Tregs.
• Treg suppressive potency remained equivalent between hUC-MSC-cocultured and control Tregs.
• Coculture significantly enhanced the expansion of all Treg subpopulations, with a greater effect on non-naive Tregs (Subtypes II and III).
• hUC-MSC coculture increased HLA-DR expression in all expanded Treg subpopulations and enhanced Foxp3+ and Helios+ percentages in non-naive Tregs.

Conclusions:

• Coculture with clinical-grade hUC-MSCs substantially boosts ex vivo Treg yield and preserves suppressive potency.
• hUC-MSC coculture modulates HLA-DR expression and enhances specific Treg subpopulations, particularly non-naive Tregs.
• These findings support the use of hUC-MSCs for improved Treg manufacturing for cell-based therapies.