Our aim in this work was to search for proteins expressed on human CD4+CD25+ thymic cells that were potentially relevant to the function and development of natural regulatory T cells.
Phage display technology has been used to identify peptide ligands in different systems and in a variety of cell types in vitro
and in vivo 
. Although antibody phage display has been useful for the identification of molecules involved in homing to the thymic endothelia 
, it was still unclear whether this technique could be appropriate for the study of new markers in natural Tregs.
In this study we used phage display to define binding sites on nTregs. The phage peptides that bound to Tregs might be exploited as a means to expand current knowledge of nTreg interactions during the development of this subtype of T cells. Besides, once these sites are clearly identified, the peptides may be used to study molecules which could improve or block the regulatory activity.
Using a peptide phage display library in a screening protocol that included subtraction and positive selection on intact viable thymocytes, we identified peptides that preferentially bound to CD4+CD25+ cells. The optimizing procedures with several rounds of panning improved the probability of obtaining phages that preferentially bound to target cells. In fact, in most cases we observed an increase in the output/input ratio during the successive rounds (). However, those data should be analyzed considering the CD4+CD25+ thymocyte purity during each step. In the first three rounds, we observed a progressive enrichment of output phages (3 fold increase), while a decrease was seen from the third to the fourth step. This decrease can be attributed to a selection using a specially enriched CD4+CD25 population in the fourth round (72% compared to 39 to 53% in the other rounds). Consequently, phages selected in the first three rounds may include those that bound to CD4+CD25+ and some that bound to CD25− cells, while phages recovered from the fourth round would be preferentially directed to our target CD4+CD25+ population. Besides, the negative selection of phages (pre-clearing) using a 90% pure CD4+CD25− population compensated for the relative low purity of CD4+CD25+ populations used for positive selection (panning).
Among the phages sequenced we selected a specific phage peptide for further analysis due to its sequence similarity to the Vitamin D receptor (VDR). This was our first choice because although Vitamin D has been extensively reported to display immunoregulatory functions in different contexts 
, its role has not been well-defined for human thymic T regs, opening an interesting area for research.
We hypothesized that the ligand recognized by the VDR phage was Vitamin D complexed to its receptor on CD4+CD25+ thymocytes, because the sequence represented by the phage peptide is located in the VDR region that interacts with the Vitamin D ligand (aa 239–246) and the structural model of the receptor revealed that the phage peptide region is located in one of the alpha helices that compose the ligand binding domain (LBD). The results from the ELISA assay showing that VDR phage was able to bind to active Vitamin D strongly supports our hypothesis. When phages were preincubated with Vitamin D we did not observe binding to VDR, confirming that the peptide, in fact, mimic the Vitamin D binding pocket in VDR () and is able to prevent Vitamin D from binding to its native receptor.
We do not know whether the identified peptide is an oligomerization domain. However, it is known that upon activation by vitamin D, VDR usually forms a heterodimer with the retinoid-X receptor and binds to hormone response elements on DNA, resulting in the expression or transrepression of specific gene products. It has also been reported that a predominant C-terminal heterodimerization domain resides between residues 382–403 in VDR sequence, about 137 aa distant from our VDR-phage. It was shown that mutations inserted in this region of the VDR sequence (Lys 382, Met-383 or Glu-385) completely disrupted the associations of VDR-RXR and VDR with other partners, and eliminated the transcriptional activity of VDR 
, indicating the importance of this region to heterodimerization. Other partners can also physiologically dimerize with VDR such as Mediator of RNA polymerase II (MED1 and MED12) 
, nuclear receptor co-repressor (NCOR1) 
and nuclear receptor co-activator (NCOA2) 
, co-activators or co-repressors that direct transcriptional initiation by the RNA polymerase II apparatus 
.Thus, although the 382–403aa domain in VDR has been shown to be important for dimerization, we cannot exclude the existence of other domains too.
The higher expression of VDR observed in CD25+ compared to CD25− cells (), if associated with a corresponding higher binding of Vitamin D to VDR in these cells, supports our isolation of VDR phage after panning in CD4+CD25+ thymocytes. Another important observation was the positive relation between Foxp3 expression and VDR expression in thymic cells (). Studies using mice in a Trinitrobenzene Sulfonic Acid (TSA) induced colitis model, treated with Vitamin D and dexamethasone, show that the combined use of Vitamin D and dexamethasone enhanced Foxp3 expression accompanied by the induction of IL-10 and TGF-beta 
. However, in vitro-generated homogenous populations of IL-10 Tregs obtained by stimulating naive CD4 T cells in the presence of the anti-inflammatory drugs Vitamin D and dexamethasone did not express high levels of Foxp3 
. Thus, although Foxp3 appears to be important for the development and function of naturally occurring CD4+CD25+ T cells, in vitro derived IL-10-secreting Tregs appear to have regulatory functions despite low levels of Foxp3.
Here, we report a high expression of VDR in thymic CD25+Foxp3+ suggesting that VDR may, indeed, be involved in the natural regulatory T cell lineage development in the thymus. Interestingly, molecules known to dimerize with VDR such as retinoic X receptor (RXR) have been shown to physically interact with Foxp3 
. The complexed VDR can dimerize with Vitamin A receptors (the retinoic acid receptor and the retinoic X receptor) and bind to Vitamin D response elements (VDrEs) in the promoters of Vitamin D-responsive genes 
. Given that Vitamin A has roles in immunoregulation that include the generation of induced Tregs 
, we hypothesize that Vitamins D and A might act synergistically in immunoregulatory pathways and possibly on natural Tregs.
An interesting question is whether the VDR phage ligand on CD4+CD25+ thymocytes is located intracellularly or on the cell surface. Although it is generally accepted that peptides presented by phage particles bind to surface molecules, investigators have reported that some phage can reach intracellular compartments by an yet unknown mechanism 
. Taking into consideration that phage can be readily internalized by cells 
, it is thus possible that the VDR phage binds directly to Vitamin D inside the cell. We did observe higher VDR intracellular expression in CD4+CD25+ thymocytes in comparison to CD4+CD25− cells (), but we were unable to detect VDR cell surface expression using the same commercial anti-VDR antibody (data not shown). We cannot be sure, however, that the anti-VDR antibody we used in our study is appropriate for cell surface expression. Nevertheless, the VDR could also (or alternatively) be expressed at the cell surface, as suggested by others 
Taken together, our results favor that Vitamin D has functions in natural Treg activity or ontogeny. Although the role of Vitamin D in natural Tregs immunobiology needs to be better defined, data from several groups showing its immunoregulatory activity on different cells 
reinforce the idea that similar functions may also be relevant for natural Tregs. The immunoregulatory role of Vitamin D has been studied in the induction of tolerogenic dendritic cells with the production of IL-10 and TGF-beta 
and in the generation of induced Tregs 
. However, in the majority of studies on T cells, Vitamin D was used in combination with other molecules, such as dexamethasone 
and IL-2 
. More recently, Vitamin D has been reported to induce and/or increase the expression of Foxp3 and CTLA-4 in purified CD4+CD25− human T cells 
. It was also suggested that Vitamin D interacts with Vitamin D Responsive Elements (VDRE) in the Foxp3 gene and enhances Foxp3 expression in CD4+ cells 
. Other data that support an immunoregulatory role for Vitamin D are derived from animal models of autoimmune diseases, in which the use of Vitamin D partially suppresses the development of experimental autoimmune encephalomyelitis, inflammatory bowel disease, and diabetes 
. The mechanisms by which Vitamin D reduces the inflammatory process in these contexts remain unclear.
In summary, our data support the notion of an immunoregulatory role for Vitamin D in natural Tregs and a functional relationship between Foxp3 and VDR in the differentiation of these cells in the thymus. This observation may shed light on possible interventional strategies for Tregs, contributing to this ongoing rich research field.