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The adoptive transfer of alloantigen-specific regulatory T cells ( allo Tregs) 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  allo Tregs 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 + CD25 hi CTV −  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,  allo Tregs were expanded up to 2,300 times the initial numbers with a purity of >95% (CD4 + CD25 hi FOXP3 + ). The resulting allospecific Tregs showed high expressions of CTLA-4, LAG-3, and CD39, indicative of a highly suppressive phenotype. Accordingly, expanded  allo Tregs efficiently suppressed T-cell proliferation in an antigen-specific manner, even in the presence of inflammatory cytokines (IFN-γ, IL-4, IL-6, or TNF-α). Unexpectedly, the long-term expansion resulted in an increased methylation of the specific demethylated region of  Foxp3 . Interestingly,  allo Tregs from both normal individuals and CKD patients maintained their immunosuppressive phenotype and function after being expanded for two additional weeks under an inflammatory microenvironment. Finally, phenotypic and functional evaluation of cryopreserved  allo Tregs demonstrated the feasibility of long-term storage and supports the potential use of this cellular product for personalized Treg therapy in transplanted patients.

frontiers in immunology

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