Article Figures & Data
Figures
- Figure 1
Chest computed tomography. The white arrow indicates a right hilar tumor of the lung and the black arrow indicates a new lesion in the right lower lobe.
- Figure 2
Serum antibody response against NY-ESO-1 in patient GO. (A) The IgG and IgM responses against recombinant NY-ESO-1 protein at a serum dilution of 1:1,600 were plotted during the course of the disease. The control recombinant protein used was RL-Akt. The clinical course and treatments shown in the box correspond to the time points. (B) Titration of serum obtained at different time points is shown. Sera from 6 healthy donors were included as a control. (C) The IgG subtype was determined using specific secondary mAbs for detection. (D) The peptide regions recognized by the antibody were determined using 30-mer NY-ESO-1 overlapping peptides.
- Figure 3
IFNγ secretion assays. MACS beads-purified CD4 and CD8 T cells (2 x 106) from PBMCs were cultured with irradiated (40 Gy) autologous CD4- and CD8-depleted PBMCs (2 x 106) as APCs in the presence of 28 overlapping 18-mer peptides and a 30-mer C-terminal peptide (OLPs) spanning the entire NY-ESO-1 protein (1 µg of each peptide/ml) in 24-well culture plates for 12 days. (A) IFNγ secretion by CD4 and CD8 T cells (1 x 105) was assayed against PFA-treated CD4- and CD8-depleted PBMCs (1 x 105) pre-pulsed with NY-ESO-1 OLPs for 30 min. (B and C) CD4 and CD8 T cells obtained in August 2005 were used on the twenty-sixth day following two stimulations. (B) IFNγ secretion by CD4 T cells (1 x 105) was assayed against the patient's EBV-transformed B cells (1 x 105) pretreated with NY-ESO-1 protein (20 µg/ml) for 24 h, and NY-ESO-1-expressing melanoma (M-1) cells (1 x 105) pretreated with IFNγ (100 U/ml) for 48 h by stimulation for 4 h. HLA class II expression on M-1 cells after IFNγ treatment is also shown. (C) IFNγ secretion by CD8 T cells (1 x 105) was assayed against the patient's PHA-stimulated CD4 T cells (T-APC) (1 x 105) transfected with NY-ESO-1 mRNA (20 µg).
- Figure 4
NY-ESO-1 regions recognized by CD4 and CD8 T cells in patient GO. MACS beads-purified CD4 and CD8 T cells (2 x 106) from PBMCs obtained in August 2005 were cultured with irradiated (40 Gy) autologous CD4- and CD8-depleted PBMCs (2 x 106) as APCs in the presence of 28 overlapping 18-mer peptides and a 30-mer C-terminal peptide (OLPs) at a concentration of 1 µg/ml of each peptide in a 24-well culture plate for 14 days. On the twenty-sixth day after two stimulations, CD4 and CD8 T cells (3 x 104) were assayed for IFNγ secretion against PFA-treated autologous CD4- and CD8-depleted PBMCs (3 x 104) pre-pulsed with the individual peptide after stimulation for 4 h. The peptide number corresponds to the individual overlapping peptides. Abbreviations: P, positive control stimulated with a mixture of OLPs; N, negative control without peptides.
- Figure 5
Restriction molecules involved in the CD4 T cell recognition of NY-ESO-1. CD4 T cell recognition of peptide 16 (aa 91-108) (A and C) and peptide 21 (aa 121-138) (B and D) was analyzed by antibody blocking (A and B) and using various EBV-B cells as APCs (C and D). CD4 T cells cultured with irradiated (40 Gy) autologous CD4- and CD8-depleted PBMCs in the presence of a mixture of OLPs for 14 days, as described in the legend of Figure 4, were assayed for IFNγ secretion against PFA-treated autologous CD4- and CD8-depleted PBMCs pre-pulsed with peptide 16 (aa 91-108) (A) and peptide 21 (aa 121-138) (B) in the presence of various mAbs (2 µg/ml) during the assay, and against PFA-treated EBV-B cells as APCs pre-pulsed with peptide 16 (aa 91-108) (1 µg/ml) (C) and peptide 21 (aa 121-138) (1 µg/ml) (D) for which HLA genotypes have been determined. IFNγ production was determined by ELISA in A and B using the supernatant after culture for 18 h and by an IFNγ secretion assay in C and D after stimulation for 4 h.
- Figure 6
NY-ESO-1-reactive CD4 and CD8 T cell frequencies. Frequency analysis of peptide-specific CD4 T cells (A) and tetramer staining of CD8 T cells (B) are shown. (A) A limited number (2 x 104) of CD4 T cells were seeded in duplicate in 96-well plates and cultured with irradiated (40 Gy) autologous CD4- and CD8-depleted PBMCs (2 x 104) as APCs in the presence of peptides 16 (aa 91-108) (1 µg/ml) (top) and 21 (aa 121-138) (1 µg/ml) (bottom) for 14 days. On the twenty-sixth day after stimulating twice, IFNγ production by the cells in each well was determined against each peptide (1 µg/ml) using autologous EBV-B cells (1 x 104) as APCs by ELISA after incubation for 18 h. An O.D. value exceeding 0.3 after subtraction of the background (without peptide) was taken as positive. The number of positive wells was 6 in both cultures. The peptide-specific CD4 T cell frequency was calculated to be 3.2 x 10-6 for both peptides. (B) Four tetramers were prepared. The patient HLA class I genotype was HLA-A*0206, A*2402, B27, B54, and Cw1. The sequence SLLMWTQC (aa 157-165) used to prepare the A*0206-tetramers lies in peptide 27 (aa 153-170) recognized by the patient's CD8 T cells, as shown in Figure 4.
- Figure 7
Flow cytometry of Foxp3+ CD25+ CD4 T regulatory cells at different time points. CD25 high and low CD4 T cells were analyzed for Foxp3 expression by intracellular staining using FACS Calibur. A normal individual was used as control.
- Figure 8
Serum TGF-β1 determined by ELISA.
Volume 9, Issue 1
