Figure 1.
Specific enrichment of CD4+CD25+ cell–bound phages.
The titer of phages recovered from each round was evaluated by a plaque-forming assay. R1–R4, 1st–4th round of panning in vitro. Cfu: colony forming units.
Table 1.
Output/input values for thymic CD4+CD25+ selection.
Table 2.
Selected peptide sequences and the corresponding human proteins containing the homologous motif.
Figure 2.
Length: of the Vitamin D Receptor; Score: punctuation score given for the alignment; Expected: e-value; Identities: number of identical amino acids between queried sequence and the corresponding amino acid sequence of the VDR protein; Positives: number of amino acids that align to the VDR protein; Gaps: spaces between amino acids that do not match; Query: VDR phage peptide sequence; Sbjct: fragment of the VDR that matches to the phage sequence. The numbers indicate the position of the amino acids in the phage peptide and in the VDR protein.
Figure 3.
ClustaW alignment of the VDR phage peptide sequence and the human VDR full-length protein sequence.
Figure 4.
Structural model of the Vitamin D Receptor associated with its ligand (Vitamin D).
Green: alpha helical sequences; gold: beta sheet; blue: structural handle, yellow: phage peptide.
Figure 5.
Binding assay with thymic CD4+CD25+ cells and CD4+CD25− cells.
Fd: wild type phage bearing no inserted peptide sequence; TU: transforming units.
Figure 6.
ELISA to evaluate the binding of the VDR phage to plate-bound Vitamin D or VDR.
Fd shows the binding of the control phage to plate-bound Vitamin D; VDRpVit shows the binding of the VDR phage to plate-bound Vitamin D; VDRpVDR shows the binding of the VDR phage to plate-bound VDR; VDRpVitVDR shows binding of the VDR phage pre-incubated with Vitamin D to plate-bound VDR. *** p = 0.001.
Figure 7.
FACS analysis of VDR expression in CD25+ and CD25− cells in the CD4+ cell compartment.
A: Dot plot showing thymocytes region; B: Dot plot showing the CD4+ gated cells; C: Dot plot showing percentage of CD25positive and negative cells in the CD4+ region; D: Histogram showing the expression of VDR in CD4+CD25+ (blue) and CD4+CD25−(red) thymocytes; E: Table showing MFI (median fluorescence intensity) of the intracellular VDR labeling of the cells.
Figure 8.
FACS analysis of VDR expression in Foxp3+ and Foxp3− cells in the CD4+CD25+ cell compartment.
(Figure 7C region). A: Dot plot showing Foxp3 region in CD4+CD25+ thymocytes; B: Histogram showing the expression of VDR in CD4+CD25+Foxp3+ (blue) and CD4+CD25+Foxp3− (red) cells; C: Table showing MFI (median fluorescence intensity) of the intracellular VDR labeling of the cells.