Isolation, expansion and functional assessment of CD4+CD25+FoxP3+ regulatory T cells and Tr1 cells from uremic patients awaiting kidney transplantation
Highlights
► We obtain CD4+CD25+FoxP3+ regulatory T cells and Tr1 cells from uremic patients. ► Regulatory T cells from uremic patients can be expanded without loss of function. ► This forms a base for adoptive regulatory T cell therapy in kidney transplantation.
Introduction
Regulatory T cells (Tregs1) play a critical role in controlling immune responses in various settings such as autoimmune diseases [1], [2], infectious diseases [3], [4], antitumor immunity [5], maternal tolerance to a fetus [6], [7] and allograft transplantation [8], [9], [10], [11]. This feat is accomplished by several mechanisms, some of which are yet to be characterized in greater detail [12], [13]. Several Treg subsets have been identified [14], [15] and among the CD4+ Tregs two major subgroups have been identified: the CD4+CD25+FoxP3+ Tregs, also known as natural Tregs (nTregs) because they develop in the thymus and are present from birth [16], and the inducible Tregs (iTregs) that are generated in the periphery from naïve T cells under tolerogenic conditions [17]. The best studied form of iTregs is the Tr1 cells, which were originally described in the mid 1990s [18], [19]. It has recently been confirmed that nTregs and Tr1 cells are indeed distinct subsets of cells with regulatory activity [20]. In the last decade the immunosuppressive properties of regulatory T cells have emerged as an attractive tool for the development of immunotherapies in a variety of disease contexts.
Here we focus on the potential use of Tregs in adoptive cell therapy to treat transplantation induced immune reactions in the setting of kidney transplantation in humans, in particular living donor kidney transplantation. Previous studies have shown higher levels of nTregs in operationally tolerant transplant patients [21], [22], [23] and several preclinical models indicate that transplantation tolerance can be induced by the transfer of nTregs [9], [24], [25], [26], [27], [28], [29]. Furthermore, the importance of nTregs to maintain peripheral tolerance is evident in patients with the IPEX syndrome (immunodysregulation, polyendocrinopathy and enteropathy, X-linked syndrome), where mutations in the FoxP3 gene cause varying degrees of enteritis and endocrinopathies [30], [31], [32], [33]. The inducible Tr1 cells also show promise for induction of transplantation tolerance [17], [18], [29], [34].
The immunosuppressive drugs used today efficiently inhibit the immune system, making solid organ transplantation possible. Unfortunately, they are associated with an increased risk of infection and certain malignancies as well as numerous drug specific side effects [35]. Albeit decreasing the risk of graft loss due to acute rejection, the number of patients experiencing chronic graft dysfunction is still high [36]. Previous studies have shown that the vasculopathy can be avoided in tolerant animals [37]; therefore, it is possible that chronic graft dysfunction can be decreased or even ameliorated with a Treg based therapy.
Section snippets
Objective
To make autologous adoptive Treg cell therapy a reality for controlling transplantation induced immune reactions Tregs need to be isolated from transplant candidates and expanded ex vivo, without loss of function. This paper focuses on the process of obtaining and functionally characterizing CD4+CD25+FoxP3+ Tregs and Tr1 cells from uremic patients awaiting kidney transplantation.
Patients
From October 2010 to March 2011 uremic patients awaiting living donor kidney transplantation, and their corresponding kidney donors, were enrolled in the study. All living kidney donor/recipient pairs were asked to participate and were given written information about the study. All patients and donors showed interest to participate, but the only pairs included were those where the transplantation date matched the capacity of available laboratory resources. This was considered a random
Isolation and expansion of CD4+CD25+FoxP3+ Tregs
Four future kidney recipients underwent leukapheresis and three had phlebotomy performed, with PBMC yields as shown in Table 2. All patients tolerated the procedure well and no adverse effects were observed. After the final step of MACS separation for CD4+CD25+FoxP3+ T cells 0.30 × 106 to 1.7 × 106 cells remained. The expansion protocol increased the number of cells 7.18–32.5 fold (21.4 ± 11.3), see Table 2.
The PBMCs from one patient (patient number 6 in Table 2) were frozen prior to MACS separation
Discussion
The present study shows that both naturally occurring regulatory T cells, with a CD4+CD25+FoxP3+ phenotype, and inducible Tr1 cells can be isolated and expanded from uremic patients without loss of function. The nTregs retain their ability to suppress an allogeneic MLR whereas the Tr1 cells produce higher levels of IL-10 when stimulated with the iDCs used for their differentiation, compared to stimulation with HLA-mismatched allogeneic iDCs.
We also show that it is logistically possible to
Acknowledgments
We thank Susanne Lindblom for her superb technical support and ever glowing enthusiasm. We also wish to express our gratitude to our study coordinator Catharina Gelin, for her administrative skills and tidiness.
The study was supported by grants from the Swedish Medical Research Council (16X-12219, K2011-65X-12219-15-6 and 2008-2205), the Juvenile Diabetes Research Foundation International and the Professor Lars-Erik Gelin Memorial Foundation.
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