IDO expressing fibroblasts promote the expansion of antigen specific regulatory T cells☆
Introduction
Immune suppression in allotransplantation is largely hindered by the toxicity of currently available immunosuppressive pharmaceutics. Suppression through this mechanism is systemic and non-specific i.e. targets the recipients’ whole immune system. Immune tolerance, on the other hand, has the benefit of being restricted to specific sites where the antigen is present. The specificity results in tolerance to the specific antigen without impairing the natural immune response to pathogens. Regulatory T cells (Tregs) are the immune system related tolerance cells. There are several subpopulations of Tregs. In this study, we focused on the classic population that is CD4+CD25+Foxp3+T cells. These cells are derived as natural Tregs directly from the thymus or can be induced from naïve T cells in the periphery. Tregs constitute 5–10% of all CD4+ T cells but only 1–10% of T cells are alloreactive T cells that are specific for alloantigens (Duan et al., 2011, Thebault et al., 2007). To develop a viable allospecific tolerance therapy an ex vivo expansion is desirable. The expansion and use of these cells would abrogate the need for immunosuppressive drugs. The specificity of Tregs to target antigens avoids generalized immune suppression and concentration of the antigen specific Tregs at the site of the allograft (Dijke et al., 2008, Saada et al., 2006).
Currently, antigen specific Treg expansion is largely achieved using dendritic cells. These cells can be induced to express the enzyme indoleamine 2,3-dioxygenase (IDO) using IFN-γ. IDO, a 42 kD monomeric protein, is expressed intracellularly in a number of cells including dendritic cells, monocytes, macrophages and fibroblasts (Jürgens et al., 2009, Daubener et al., 2009). It rapidly oxidizes l-tryptophan, an essential amino acid, to N-formylkynurenine which then deformylates to form kynurenine (Sugimoto et al. 2006). Kynurenine can be further metabolized along the kynurenine pathway into kynurenine metabolites (Munn et al., 2005, Sugimoto et al., 2006). IDO has a high affinity for l-tryptophan (Km ∼ 0.02 mM) and rapid catabolism results in a local tissue microenvironment deficient in this essential amino acid (King and Thomas 2007). In 1998, Munn and Mellor showed that IDO is important in immune regulation and generation of immune privilege during pregnancy (Munn et al. 1998). Inhibition of IDO expression in trophoblast cells of the placenta using 1-methyl tryptophan resulted in spontaneous abortion in a murine model. Following this initial discovery, it has become clear that tryptophan deficiency induces T cells to transition to a state of anergy, apoptosis or transdifferentiation into regulatory T cells (Chung et al., 2009, Jürgens et al., 2009, Katz et al., 2008).
As mentioned previously, IDO is also expressed by fibroblasts in response to IFN-γ. In the skin, depletion of tryptophan in the environment has a role in inhibiting the proliferation of intra-cellular bacteria and parasites. Fibroblasts are non-professional antigen presenting cells that can be induced to express MHC class II and co-stimulatory molecules (Saada et al. 2006). Thus it is possible that fibroblasts, like dendritic cells be able to expand a population of antigen specific Tregs. Induction of IDO in fibroblasts will additionally support the survival of Tregs over effector cells in a co-culture system (Dai and Gupta, 1990, Pfefferkorn and Rebhun, 1986). The survival of fibroblasts, importantly is not affected in this environment (Forouzandeh et al. 2008b). In contrast to DCs, however, fibroblasts are easily isolated from a skin biopsy, are robust and can be maintained in culture over a prolonged number of passages and additionally can be defrosted without much impact on their functionality. DCs must be isolated as fresh primary cells, the numbers achieved on isolation are much smaller than those that can be cultivated from fibroblasts and they do not function well after freezing. In this study, we demonstrate that fibroblasts expressing IDO, like DCs can promote expansion of an antigen specific Treg population.
Section snippets
Mice
Male BALB/c, FVB/N and C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA). Mice were housed in ventilated cages in sets of 4 with food and water available ad libitum, as per federal and institutional ethical recommendations. All experiments were carried out according to standard operating procedures approved by the UBC animal ethics committee and by recipients holding full animal husbandry certification.
Co-culture of fibroblasts with splenocytes
Spleens from male BALB/c mice were removed under sterile
MHC II and IDO genes expression was confirmed in the co-cultured fibroblasts
To confirm the IFN-γ activity in induction of IDO expression in dermal fibroblasts, the level of kynurenine, as an index for IDO activity, in conditioned medium of treated and untreated cells was evaluated. The result showed a significantly higher level (4.77 ± 0.58 μmol/L vs. 0.08 ± 0.04 μmol/L, p-value < 0.001) of kynurenine in conditioned medium from the IFN-γ treated fibroblasts relative to that of control (Fig. 1A). As we wanted to test the efficacy of the conditioned medium of IDO expressing
Discussion
This study demonstrates a novel role of IDO- expressing fibroblasts in expanding an antigen specific Treg population. Induction and expansion of antigen specific Tregs by IDO expressing dendritic cells (DCs) is a well-known fact (Jürgens et al., 2009, Onodera et al., 2009). However, it is very burdensome to isolate and maintain sufficient DCs to produce large quantities of Tregs. This obstacle has significantly hindered feasibility of this promising tolerance induction strategy. We suggest that
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgements
This research was funded by Transplant Research Foundation of British Columbia and the Canadian Institute of Health Research (AG) as well as a generous support from Spectra Energy. T.A.C. received funding from CIHR Transplant Training Program and WorkSafe BC Research Training Award. R.B.J. received postdoctoral fellowship award from Juvenile Diabetes Research Foundation.
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Contract grant sponsor: Canadian Institutes of Health Research, Transplant Research Foundation of British Columbia.