Peptide Epitopes from the Wilms' Tumor 1 Oncoprotein Stimulate CD4+ and CD8+ T Cells That Recognize and Kill Human Malignant Mesothelioma Tumor Cells

Rena J. May; Tao Dao; Javier Pinilla-Ibarz; Tatyana Korontsvit; Victoriya Zakhaleva; Rong H. Zhang; Peter Maslak; David A. Scheinberg

doi:10.1158/1078-0432.CCR-07-0708

DOI: 10.1158/1078-0432.CCR-07-0708 Published August 2007

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Fig. 1.
IFN-γ ELISPOT with WT1DR peptides 328, 423, 122, and 1222A1. A, CD3⁺ T cells from an HLA-DRB1*1001/1501 donor were stimulated twice with either peptide WT1DR 328 or WT1DR 423 as described in Materials and Methods. Stimulated T cells were challenged in an IFN-γ ELISPOT assay with the following: unchallenged control (gray columns), CD14⁺ cells pulsed with stimulating peptide (either WT1DR 328 or WT1DR 423; black columns), CD14⁺ cells pulsed with irrelevant CD4⁺ peptide epitope (RAS; white columns), and unpulsed CD14⁺ cells (hatched columns). *, P < 0.05, compared with controls. Y axis, number of spots per 1 × 10⁵ CD3⁺ T cells; X axis, peptide used for T-cell stimulations. B, CD4⁺ T cells from an HLA-DRB1*1001/1501 donor were stimulated twice with either peptide WT1DR 328 or WT1DR 423 as described in Materials and Methods. Stimulated T cells were challenged in an IFN-γ ELISPOT assay with the following: CD14⁺ cells pulsed with stimulating peptide (either WT1DR 328 or WT1DR 423; black columns), CD14⁺ cells pulsed with stimulating peptide (either WT1DR 328 or WT1DR 423) and incubated with anti-HLA-DR blocking antibody (gray hatched columns), CD14⁺ cells pulsed with irrelevant CD4⁺ peptide epitope (CML b2a2; white columns), CD14⁺ cells pulsed with irrelevant CD4⁺ peptide epitope (CML b2a2) and incubated with anti-HLA-DR blocking antibody (checkerboard columns), and unpulsed CD14⁺ cells (black hatched columns). *, P < 0.04, compared with irrelevant peptide controls and compared with CD14⁺ T cells in the presence of anti-HLA-DR monoclonal antibody. Y axis, number of spots per 1 × 10⁵ CD4⁺ T cells; X axis, peptide used for T-cell stimulations. C, CD4⁺ T cells from an HLA-DRB1*1501 donor were stimulated twice with either peptide WT1DR 122 or WT1DR 122A1 as described in Materials and Methods. Stimulated T cells were challenged in an IFN-γ ELISPOT assay with the following: unpulsed CD14⁺ cells (light gray columns), CD14⁺ cells pulsed with irrelevant CD4 peptide epitope (CML b2a2; white columns), CD14⁺ cells pulsed with irrelevant CD4 peptide epitope (CML b2a2) and incubated with anti-HLA-DR blocking antibody (dotted columns), CD14⁺ cells pulsed with peptide WT1DR 122 (dark gray columns), CD14⁺ cells pulsed with peptide WT1DR 122 and incubated with anti-HLA-DR blocking antibody (dark gray hatched columns), CD14⁺ cells pulsed with peptide WT1DR 122A1 (black columns), and CD14⁺ cells pulsed with peptide WT1DR 122A1 and incubated with anti-HLA-DR blocking antibody (black hatched columns). *, P < 0.05, compared with irrelevant peptide controls and compared with CD14⁺ T cells in the presence of peptide and anti-HLA-DR monoclonal antibody. Y axis, number of spots per 1 × 10⁵ CD4⁺ T cells; X axis, peptide used for T-cell stimulations.
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Fig. 2.
Quantitative reverse transcription-PCR. Relative WT1 expression levels in a variety of hematopoietic and mesothelioma cell lines. WT1 levels are shown as relative values compared with the human leukemia cell line K562, which is defined as 1.0 as described in ref. 28.
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Fig. 3.
Processing and presentation of WT1DR peptides. A and B, cross-priming experiments. A, CD3⁺ T cells from an HLA-A0201/301 DRB1*1301/1302 healthy donor were stimulated with autologous dendritic cells (DC) previously incubated with 697 tumor lysates. 697 is a WT1⁺ leukemia cell line. Stimulated T cells were challenged in an IFN-γ ELISPOT assay with autologous dendritic cells previously incubated with either 697 tumor lysate, individual WT1 peptides, control peptides, or unpulsed dendritic cells, as indicated on the X axis. Hatched columns, background level of spots from autologous dendritic cells incubated in the absence of T cells. *, P < 0.05, compared with control peptides. Y axis, number of spots per 1 × 10⁵ CD3⁺ cells. B, CD3⁺ T cells from an HLA-A0201/101 DRB1*0301/1601 healthy donor were stimulated with autologous dendritic cells previously incubated with tumor lysates from either JMN, a WT1⁺ mesothelioma cell line (black columns), or MeWo, a WT1⁻ melanoma cell line (white columns). Stimulated T cells were challenged in an IFN-γ ELISPOT assay with autologous dendritic cells previously incubated with either JMN or MeWo tumor lysates, individual WT1DR peptides, or control class II peptide, as indicated on the X axis. Hatched columns, background level of spots from autologous dendritic cells incubated in the absence of T cells. *, P < 0.05, compared with control peptides. Y axis, number of spots per 1 × 10⁵ CD3⁺ cells. C and D, CD3⁺ IFN-γ ELISPOT against mesothelioma cell lines. C, total PBMCs from an HLA-DRB1*13XX donor were stimulated twice with the different WT1DR peptides as described in Materials and Methods. Stimulated T cells were challenged in an IFN-γ ELISPOT assay with the following: mesothelioma H-Meso1A cell line (WT1⁺, HLA-DRB1*1301; black columns) and control melanoma MeWo cell line (WT1⁻, HLA-DRB1*15XX; gray columns). *, P ≤ 0.01, compared with MeWo controls. Y axis, number of spots per 2 × 10⁵ PBMCs; X axis, peptide used for T-cell stimulation. D, CD3⁺ T cells from an HLA-A0201/DRB1*1501 donor were stimulated twice with WT1DR 122A1 as described in Materials and Methods. Stimulated T cells were then challenged in an IFN-γ ELISPOT assay with the following target cells: JMN, an A0201/DRB1*1505 WT1⁺ mesothelioma cell line, or MeWo, an A0201/DRB1*15XX WT1⁻ melanoma cell line. The target cells were either pulsed with WT1DR 122A1 (black columns) or not pulsed (gray columns). *, P < 0.05, compared with the unpulsed MeWo target cell. Y axis, number of spots per 1 × 10⁵ CD3⁺ T cells; X axis, different cell lines used as target cells.
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Fig. 4.
WT1DR peptide 122 and 122A1 stimulate CD8⁺ T-cell responses. A, CD3⁺ T cells from an HLA-A0201/DRB1*1401 donor were stimulated twice with WT1DR 122 as described in Materials and Methods. Stimulated T cells were challenged in an IFN-γ ELISPOT assay with autologous CD14⁺ cells in the presence of different peptides. Y axis, number of spots per 1 × 10⁵ CD3⁺ cells; X axis, different test peptides used in the ELISPOT. B, CD3⁺ T cells from an HLA-A0201/DRB1*1401 donor were stimulated twice with WT1DR 122A1 as described in Materials and Methods. Stimulated T cells were challenged in an IFN-γ ELISPOT assay with control melanoma cell line MeWo (A0201/DRB1*15XX, WT1⁻) in the presence of different peptides. *, P < 0.05, compared with no peptide controls. Y axis, number of spots per 1 × 10⁵ CD3⁺ cells; X axis, different test peptides used in the ELISPOT. C, CD3⁺ T cells from an HLA-A0201/DRB1*0101/15XX donor were stimulated twice with WT1DR 122A1 as described in Materials and Methods. After two rounds of stimulation, CD8⁺ T cells were isolated by negative selection and used as effector cells in a ⁵¹Cr release cytotoxicity assay as described in Materials and Methods. CD8⁺ T cells were incubated with various radiolabeled target cells [pulsed or unpulsed 697 (A0201⁺, WT1⁺) or SKLY16 (A0201⁺, WT1⁻)] at three different E:T ratios: 100:1 (gray columns), 30:1 (black columns), and 10:1 (white columns). Y axis, percentage of cytotoxicity; X axis, different target cell conditions. *, P < 0.05, compared with SKLY16 controls at the same E:T ratio. D, CD3⁺ T cells from an HLA-A0201/DRB1*0101/15XX donor were stimulated twice with WT1DR 122A1 as described in Materials and Methods. After two rounds of stimulation, CD8⁺ T cells were isolated by negative selection and used as effector cells in a ⁵¹Cr release cytotoxicity assay as described in Materials and Methods. CD8⁺ T cells were incubated with radiolabeled JMN (A0201⁺, WT1⁺; black line) or MeWo (A0201⁺, WT1⁻, gray line) target cells at four different E:T ratios. Y axis, percentage of cytotoxicity; X axis, different E:T ratios. P < 0.001, compared with MeWo controls.

Tables

Figures

Table 1.

Peptides from WT1 that are predicted to bind to HLA-DRB molecules

Name	Sequence	Score*
Name	Sequence			*DRB10101**	*DRB10301**	*DRB10401**	*DRB10701**	*DRB11101**	*DRB11501**
423	RSDELVRHHNMHQRNMTKL	15	17	20	14	10	24
328	PGCNKRYFKLSHLQMHSRKHTG^†	28	11	28	18	25	20
122	SGQARMFPNAPYLPSCLES^‡	22	18	22	16	16	18
122A1	SGQAYMFPNAPYLPSCLES^§	27	17	22	18	16	18
Frequency in Caucasian population (49)			18.5	17.7	23.6	26.2	17.0	19.9

↵* SYFPEITHI prediction software available at http://www.syfpeithi.de.
↵† Peptide encompasses the sequence reported by ref. 29 (WT1 amino acids 331-345).
↵‡ Peptide encompasses the sequence reported by Kobayashi et al. (WT1 amino acids 124-138; ref. 31).
↵§ Residue Y in bold represents a modification from the native 122 sequence.

Table 2.

Summary of in vitro stimulation experiment and IFN-γ ELISPOT results

Experiment no.	WT1DR 328			WT1DR 423			WT1DR 122
Experiment no.		Donor HLADRB*	Response^†	Donor HLADRB	Response	Donor HLADRB	Response	Donor HLADRB	Response
1	1001/1501	+++	1001/1501	+++	1401/15XX	—	1401/15XX	+ (heteroclitic)^‡
2	301/901	+++	101/1201	—	1401	+	1401	++ (heteroclitic)
3	1001/1501	++	1001/1501	—	1401	+	1401	+ (heteroclitic)
4	407/1302	NA^§	407/1302	NA	104/1104	—	104/1104	+++ (heteroclitic)
5	407/1302	+++	407/1302	+	405/1302	—	301/1601	NA
6	1001/1501	—	1001/1501	—	301/1601	++	405/1302	—
7	407/1302	—	407/1302	—	1401/15XX	+++	15XX	+++ (heteroclitic)
8	1001/1501	++	1001/1501	—	15XX	+	422/13XX	+++ (heteroclitic)
9	407/1302	++	407/1302	+	1401/15XX	+++	422/13XX	++ (heteroclitic)
10	701/1202	++	701/1202	—			301/1601	—
11	1401/15XX	++	1401/15XX	—			1401/15XX	+
12	407/1302	+++	422/13XX	—			1401/15XX	+
13	422/13XX	+	422/13XX	+++			15XX	+++ (heteroclitic)
14	422/13XX	+++	1001/1501	+++
15	1001/1501	+++
No. donors	6	80%^∥	6	35.7%^∥	6	66.6%^∥	7	76.9%^∥

↵* Healthy donor CD3⁺/CD4⁺ T cells were stimulated twice with individual WT1 peptide as described in Materials and Methods. Stimulated T cells were challenged in an IFN-γ ELISPOT.
↵† ELISPOT results comparing T cell reactivity with autologous CD14⁺ cells pulsed with stimulating peptide versus autologous CD14⁺ cells pulsed with irrelevant peptide. −, negative results; +, two times background; ++, three times background; +++, four times background.
↵‡ T cells stimulated with WT1DR peptide 122A1 recognized CD14⁺ cells pulsed with stimulating peptide and CD14⁺ cells pulsed with WT1DR peptide 122.
↵§ Experiment failed.
↵∥ Percentage of experiments with a significant positive results (P ≤ 0.05).