IFN-γ ELISPOT by CD3, CD4 or CD8 T cells stimulated with cyclin D1-DR1 or D1-1 peptides.

<p>(A) CD3 or CD4 T cells from a healthy donor (HLA-A0201 and DR.B1 0104/1104) were stimulated twice with human cyclin D1-derived peptides DR-1, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006730#s2" target="_blank">Materials and Methods</a>. (B) CD3 or CD8 T cells from a healthy donor (HLA-A0201 and HLA-DR.B1 0801/0901) were stimulated three times with human cyclin D1-derived peptides D1-1. Stimulated T cells were challenged in IFN-γ ELISPOT assay with the stimulating peptide DR-1 or D1-1. The controls are: un-pulsed CD14+ cell as APCs, irrelevant peptides JAK2-DR for HLA-DR.B1 and EW for HLA-A0201. The results represent the average spots in triplicate cultures +/− SD. The figures show one representative data from multiple similar experiments.</p

IFN-γ ELISPOT by CD4+CD25+ and CD4+CD25- T cells stimulated with cyclin D1-DR1 peptide.

<p>CD4+ (A) or CD4+CD25- T cells (B) from healthy donors were stimulated three times with DR-1 peptide, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006730#s2" target="_blank">Materials and Methods</a>. Stimulated T cells were challenged in IFN-γ ELISPOT assay with the stimulating peptide DR-1 or controls on CD14+ cells, B cells or MDA-MB-231 cells. Antibody blocking was performed by adding the anti-HLA-DR and isotype control mAb (50 ug/ml) in the cultures. The controls are: CD4 T cells cultures with un-pulsed CD14+ cells, irrelevant peptides JAK2-DR for HLA-DR.B1, and allogenic B cells from a donor who has matching HLA-DR.B1*0701 with MDA-MB-231 cells. The results represent the average spots in triplicate cultures +/− SD. The figures show one representative data from three similar experiments.</p

The expression of cyclin D1 and HLA molecules determined by flow cytometry.

<p>Cyclin D1 expression on mature DCs and MDA-MB-231 cell line was determined by intracellular protein staining using Cytofix/Cytoperm kit (A). The cells were stained with the mAb to human cyclin D1, or isotype control mouse IgG1, then were followed by the secondary antibody goat anti-mouse Ig (Fab)2 conjugated to FITC. Solid and dash lines are the DCs and MDA-MB-231 cells stained with isotype controls, respectively. The dash-dot-dash and dotted lines show the cyclin D1 expression on DCs and MDA-MB-231 cells, respectively. HLA-A0201 (B) or HLA-DR (C) expression on MBA-MB-231 was determined by surface staining of the cells with mAbs to HLA-A2 or HLA-DR, before and after treatment with IFN-γ. Dash and solid lines show the isotype controls before and after IFN-γ treatment, respectively. The dash-dot-dash and dot lines show HLA-A0201 expression before or after IFN-γ treatment (B). Similarly, the dash and solid lines show the isotype controls staining before and after IFN-γ treatment; the dot and dash-dot-dash lines show the HLA-DR expression before or after IFN-γ treatment (C).</p

Peptides from human cyclin D1-a that are predicted to bind to HLA-DR.B1 and embedded short peptides (in italics) that bind to HLA-A0201 molecules.

*<p>SYFPEITHI prediction software was used for HLA-DR.B1-binding prediction. Each epitope has different binding affinity to various DR.B1 subtypes, which was summarized as the range of binding scores. RANKPEP prediction software was used for HLA-A0201 prediction and C-terminal cleavage.</p

Induction of cytotoxicity by CD3 and CD4 T cells stimulated with DR-1 peptide.

<p>CD3 T cells from a HLA-A0201 donor were stimulated with DR-1 peptide for three times, and the cytotoxicity of the cells was measured by ⁵¹Cr-release assay against autologous DCs pulsed with or without indicated peptides (A). Similarly, the purified CD4 T cells from a HLA-A0201 and HLA-DR.B1-0701 donor was stimulated with DR-1 peptide and their cytotoxicity was measured by ⁵¹C-release assay against autologous DCs pulsed with or without indicated peptides, and MDA-MB-231 cell line (B). Antibody blocking was performed by pre-incubating target cells with anti-HLA-DR and isotype-matching irrelevant mAb (50 ug/ml) for 30 minutes before adding to the co-culture with effector cells at an E: T ratio of 100∶1 (C). The figure shows representative data from multiple similar experiments. All data points are the mean of triplicate microwell cultures.</p

IFN-γ ELISPOT by CD3 T cells stimulated with cyclin D1 peptides.

<p>CD3 T cells from healthy donors with HLA-A0201 and various HLA-DR.B1 haplotypes were stimulated three times with human cyclin D1-derived peptides DR-1 (A), DR-2 (B), DR-3 (C), DR-4 (D), DR-5 (E) or D1-1 (F), as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006730#s2" target="_blank">Materials and Methods</a>. Stimulated CD3 T cells were challenged in IFN-γ ELISPOT assay with the stimulating HLA-DR.B1 class II peptides and the HLA-A0201 class I peptides that were imbedded within the class II peptides, as indicated. Controls are: un-pulsed CD14+ cells, irrelevant peptides JAK2-DR for HLA-DR.B1 and EW for HLA-A0201. The results represent the average spots in triplicate cultures plus/minus standard deviation (SD). The figures show one representative experiment from multiple similar experiments.</p