Highly Purified Alloantigen-Specific Tregs From Healthy and Chronic Kidney Disease Patients Can Be Long-Term Expanded, Maintaining a Suppressive Phenotype and Function in the Presence of Inflammatory Cytokines

The adoptive transfer of alloantigen-specific regulatory T cells (alloTregs) has been proposed as a therapeutic alternative in kidney transplant recipients to the use of lifelong immunosuppressive drugs that cause serious side effects. However, the clinical application of alloTregs has been limited due to their low frequency in peripheral blood and the scarce development of efficient protocols to ensure their purity, expansion, and stability. Here, we describe a new experimental protocol that allows the long-term expansion of highly purified allospecific natural Tregs (nTregs) from both healthy controls and chronic kidney disease (CKD) patients, which maintain their phenotype and suppressive function under inflammatory conditions. Firstly, we co-cultured CellTrace Violet (CTV)-labeled Tregs from CKD patients or healthy individuals with allogeneic monocyte-derived dendritic cells in the presence of interleukin 2 (IL-2) and retinoic acid. Then, proliferating CD4+CD25hiCTV− Tregs (allospecific) were sorted by fluorescence-activated cell sorting (FACS) and polyclonally expanded with anti-CD3/CD28-coated beads in the presence of transforming growth factor beta (TGF-β), IL-2, and rapamycin. After 4 weeks, alloTregs were expanded up to 2,300 times the initial numbers with a purity of >95% (CD4+CD25hiFOXP3+). The resulting allospecific Tregs showed high expressions of CTLA-4, LAG-3, and CD39, indicative of a highly suppressive phenotype. Accordingly, expanded alloTregs efficie...
Source: Frontiers in Immunology - Category: Allergy & Immunology Source Type: research