Abstract
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 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, alloTregs 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 alloTregs demonstrated the feasibility of long-term storage and supports the potential use of this cellular product for personalized Treg therapy in transplanted patients.
Highlights
Kidney transplantation is currently the therapy of choice for most causes of chronic kidney disease (CKD) [1]
For the initial expansion of allospecific Tregs, we used the combination of retinoic acid (RA) plus interleukin 2 (IL-2) in the co-cultures, which led to 44.8 ± 9.1% of viable proliferating cells, being CD4+CD25+CellTrace Violet (CTV)− Tregs (Figure S5A)
To increase the yield of alloTregs, FACS-sorted CD4+CD25hiCD127− Tregs were expanded in the presence of allogeneic dendritic cell (DC) plus RA and IL-2, which increased the numbers of purified alloTregs compared to previous reports using rapamycin in primary co-cultures (Figure S5A) [18]
Summary
Kidney transplantation is currently the therapy of choice for most causes of chronic kidney disease (CKD) [1]. Kidney transplant (KT) patients are treated with immunosuppressive drugs that reduce the rates of renal acute rejection and increase patients’ survival [2]. It is still necessary to develop new therapies that induce specific allograft tolerance. Tregs are characterized by the expression of FOXP3, a transcription factor that regulates their immunosuppressive function [4]. Studies in mouse models have demonstrated that Tregs are essential to inducing specific KT tolerance by the inhibition of effector T cells and modulating dendritic cell function [5]. KT patients who developed clinical operational tolerance exhibit a significant increase in the number of circulating FOXP3+ Tregs [6, 7]. The clinical application of Tregs has been extensively studied as an approach for the induction of allotransplant tolerance [8]
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