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Correlation of high and decreased NY-ESO-1 immunity to spontaneous regression and subsequent recurrence in a lung cancer patient

Midori Isobe, Shingo Eikawa, Akiko Uenaka, Yoichi Nakamura, Tetsuo Kanda, Shigeru Kohno, Kiyotaka Kuzushima and Eiichi Nakayama
Midori Isobe
1Department of Immunology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
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Shingo Eikawa
1Department of Immunology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
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Akiko Uenaka
1Department of Immunology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
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Yoichi Nakamura
2Department of Medicine, Nagasaki University School of Medicine, Nagasaki, Japan
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Tetsuo Kanda
3Department of Internal Medicine, Goto Central Hospital, Goto, Japan
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Shigeru Kohno
2Department of Medicine, Nagasaki University School of Medicine, Nagasaki, Japan
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Kiyotaka Kuzushima
4Department of Immunology, Aichi Cancer Center, Nagoya, Japan
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Eiichi Nakayama
1Department of Immunology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
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DOI:  Published January 2009
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    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.

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    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.

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    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).

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    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.

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    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.

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    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.

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    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.

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    Figure 8

    Serum TGF-β1 determined by ELISA.

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Cancer Immunity Archive: 9 (1)
January 2009
Volume 9, Issue 1
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Correlation of high and decreased NY-ESO-1 immunity to spontaneous regression and subsequent recurrence in a lung cancer patient
Midori Isobe, Shingo Eikawa, Akiko Uenaka, Yoichi Nakamura, Tetsuo Kanda, Shigeru Kohno, Kiyotaka Kuzushima and Eiichi Nakayama
Cancer Immun January 1 2009 (9) (1) 8;

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Correlation of high and decreased NY-ESO-1 immunity to spontaneous regression and subsequent recurrence in a lung cancer patient
Midori Isobe, Shingo Eikawa, Akiko Uenaka, Yoichi Nakamura, Tetsuo Kanda, Shigeru Kohno, Kiyotaka Kuzushima and Eiichi Nakayama
Cancer Immun January 1 2009 (9) (1) 8;
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