Wilms tumor gene 1 product (WT1) has been recognized as an attractive target antigen of immunotherapy for various malignancies including leukemia. Because tumor-associated antigen-specific CD4+ T lymphocytes undoubtedly play an important role in the induction of an antitumor immune response, we attempted to generate WT1-specific CD4+ T lymphocytes in vitro and examined their antileukemia functions. A CD4+ T-cell line, designated NIK-1, which proliferated and produced Th1 cytokines specifically in response to stimulation with the WT1-derived peptide, WT1337-347 LSHLQMHSRKH, in an HLA-DP5-restriced manner was established. NIK-1 exhibited cytotoxicity against HLA-DP5-positive, WT1-expressing leukemia cells but did not lyse HLA-DP5-negative, WT1-expressing leukemia cells or HLA-DP5-positive, WT1-negative cells. NIK-1 did not inhibit colony formation by normal bone marrow cells of HLA-DP5-positive individuals. This is the first report to describe WT1-specific and HLA class II-restricted CD4+ T lymphocytes possessing direct cytotoxic activity against leukemia cells. (Blood. 2005;106: 1415-1418)

Because Wilms tumor gene 1 product (WT1) is expressed in most cases of acute leukemia but not in normal tissues, it would be an attractive target antigen for immunotherapy against various malignancies including leukemia.1-4  Recently, we and other investigators have succeeded in generating CD8+ cytotoxic T lymphocytes (CTLs) that recognize WT1-derived peptides in vitro.5-9  These WT1-specific CTLs efficiently lysed leukemia cells and solid tumor cells, but not normal cells, in an HLA class I-restricted manner. On the basis of these findings, clinical trials of cancer vaccine using WT1 peptides have been initiated.10,11 

Increasing evidence from both murine and human studies indicates that tumor-associated antigen-specific CD4+ T lymphocytes play a central role in orchestrating the host immune response against malignancies and infectious diseases.12  Because identification of epitopes on WT1 recognized by CD4+ T lymphocytes is essential for development of effective cellular immunotherapy for malignaicies targeting WT1, we attempted to generate WT1-derived peptide-specific CD4+ T lymphocytes and examined their antileukemia functions.

Approval for the present study was obtained from the Institutional Review Board of Ehime University School of Medicine. Informed consent was obtained according to the Declaration of Helsinki.

On the basis of the amino acid sequence of WT1, a comprehensive panel of 43 20-mer peptides with 10 overlapping amino acids were synthesized. The WT1 peptide-specific CD4+ T-cell lines were generated as reported previously.13  Briefly, peripheral blood mononuclear cells (PBMCs) were stimulated 3 times with synthetic peptides at a concentration of 10 μg/mL. Cells showing a positive response to a WT1 peptide were cultured continuously in interleukin-2 (IL-2)-containing culture medium, and mitomycin C-treated autologous PBMCs and WT1 peptide were added to the wells every 1 to 2 weeks.

Chromium-51 release cytotoxicity assays were performed as described previously.14  In some experiments, the target cells were incubated with an anti-HLA-DR monoclonal antibody (mAb) (L243), an anti-HLA-DQ mAb (HU-11), or anti-HLA-DP mAb (B7/21) at an optimal concentration for 30 minutes before adding the effector cells. Cold-target inhibition assays were performed as described previously.15 

WT1 mRNA expression levels in cells were determined by quantitative reverse-transcription polymerase chain reaction (PCR) and calculated relative to that in the human leukemia cell line K562 as described previously.16 

The effect of WT1-specific T lymphocytes on the growth of normal bone marrow cells was examined by performing the colony-forming assays as described previously.5 

A CD4+ T-cell line, designated NIK-1, which proliferated specifically in response to stimulation with one of the 20-mer WT1 peptides (WT1336-355 KLSHLQMHSRKHTGEKPYQC) was established. More than 99% of NIK-1 cells were CD3+CD4+CD8-. NIK-1 appeared to produce large amounts of T helper-1 (Th1) cytokines, such as interferon-γ, IL-2, and IL-12, upon stimulation with WT1 peptide in the presence of autologous PBMCs (data not shown). In addition to the proliferative response, NIK-1 showed strong cytotoxicity against a WT1 peptide-loaded autologous B-lymphoblastoid cell line (LCL) (Table 1). The restriction element of NIK-1 appeared to be HLA-DP, because the cytotoxicity and proliferative response of NIK-1 against WT1 peptide were significantly inhibited by adding anti-HLA-DP mAb. Because the donor was an HLA-DP5 homozygote, the cytotoxicity of NIK-1 against WT1 peptide-loaded HLA-DP5 gene-transfected L cells was examined. As expected, NIK-1 exerted cytotoxicity against WT1 peptide-loaded HLA-DP5-positive L cells but not against HLA-DP9+ L cells (Table 2). In addition, NIK-1 showed cytotoxicity against WT1 peptide-loaded HLA-DP5-positive but not HLA-DP5-negative allogeneic cells (Table 1), demonstrating that NIK-1 is the HLA-DP5-restricted CD4+ T-cell line.

Table 1.

WT1-specific and HLA-DP5-restricted cytotoxicity by NIK-1: experiment 1



% cytotoxicity, at E/T ratio
Target cells
10:1 ratio
5:1 ratio
2.5:1 ratio
Auto LCL (HLA-DP5-positive)    
Without WT1 peptide   10.3   6.3   3.1  
With WT1 peptide   70.2   63.2   49.1  
Allo LCL no. 1 (HLA-DP5-positive)    
Without WT1 peptide   8.9   7.3   3.4  
With WT1 peptide   73.9   62.1   50.1  
Allo LCL no. 2 (HLA-DP5-positive)    
Without WT1 peptide   5.3   5.1   4.1  
With WT1 peptide   63.2   56.1   47.3  
Allo LCL no. 3 (HLA-DP5-negative)    
Without WT1 peptide   1.0   0.5   0.0  
With WT1 peptide   2.1   0.8   0.1  
Allo LCL no. 4 (HLA-DP5-negative)    
Without WT1 peptide   2.1   0.0   0.1  
With WT1 peptide
 
4.1
 
0.8
 
0.0
 


% cytotoxicity, at E/T ratio
Target cells
10:1 ratio
5:1 ratio
2.5:1 ratio
Auto LCL (HLA-DP5-positive)    
Without WT1 peptide   10.3   6.3   3.1  
With WT1 peptide   70.2   63.2   49.1  
Allo LCL no. 1 (HLA-DP5-positive)    
Without WT1 peptide   8.9   7.3   3.4  
With WT1 peptide   73.9   62.1   50.1  
Allo LCL no. 2 (HLA-DP5-positive)    
Without WT1 peptide   5.3   5.1   4.1  
With WT1 peptide   63.2   56.1   47.3  
Allo LCL no. 3 (HLA-DP5-negative)    
Without WT1 peptide   1.0   0.5   0.0  
With WT1 peptide   2.1   0.8   0.1  
Allo LCL no. 4 (HLA-DP5-negative)    
Without WT1 peptide   2.1   0.0   0.1  
With WT1 peptide
 
4.1
 
0.8
 
0.0
 

Cytotoxicity of NIK-1 against autologous (auto) and various allogeneic (allo) LCL loaded or unloaded with WT1 peptide. E/T ratio indicates effector-target ratio.

Table 2.

WT1-specific and HLA-DP5-restricted cytotoxicity by NIK-1: experiment 2


Target cells

% cytotoxicity, at 10:1 E/T ratio
Auto LCL (HLA-DP5-positive)  
Without WT1 peptide   10.5  
With WT1 peptide   63.6  
With WT1 peptide and anti-HLA-DR mAb   59.9  
With WT1 peptide and anti-HLA-DQ mAb   62.5  
With WT1 peptide and anti-HLA-DP mAb   11.6  
L-DP5 (HLA-DP5-positive)  
Without WT1 peptide   5.7  
With WT1 peptide   68.3  
With WT1 peptide and anti-HLA-DR mAb   62.6  
With WT1 peptide and anti-HLA-DQ mAb   64.7  
With WT1 peptide and anti-HLA-DP mAb   8.9  
L-DP9 (HLA-DP5-positive)  
Without WT1 peptide   2.5  
With WT1 peptide
 
4.2
 

Target cells

% cytotoxicity, at 10:1 E/T ratio
Auto LCL (HLA-DP5-positive)  
Without WT1 peptide   10.5  
With WT1 peptide   63.6  
With WT1 peptide and anti-HLA-DR mAb   59.9  
With WT1 peptide and anti-HLA-DQ mAb   62.5  
With WT1 peptide and anti-HLA-DP mAb   11.6  
L-DP5 (HLA-DP5-positive)  
Without WT1 peptide   5.7  
With WT1 peptide   68.3  
With WT1 peptide and anti-HLA-DR mAb   62.6  
With WT1 peptide and anti-HLA-DQ mAb   64.7  
With WT1 peptide and anti-HLA-DP mAb   8.9  
L-DP9 (HLA-DP5-positive)  
Without WT1 peptide   2.5  
With WT1 peptide
 
4.2
 

Cytotoxicity of NIK-1 against autologous LCL, HLA-DP5 gene-transduced L cells, and HLA-DP9 gene-transduced L cells loaded or unloaded with WT1 peptide in the presence or absence of anti-HLA-DR, anti-HLA-DQ, or anti-HLA-DP mAb. E/T ratio indicates effector-target ratio.

We next examined the fine epitope on WT1 recognized by NIK-1. Experiments using deletion peptides clearly demonstrated that the minimal amino acid sequence recognized by NIK-1 is WT1337-347 LSHLQMHSRKH (Table 3).

Table 3.

WT1-specific and HLA-DP5-restricted cytotoxicity by NIK-1: experiment 3


WT1 peptide loaded

% cytotoxicity, at 10:1 E/T ratio
None   3.5  
KLSHLQMHSRKHTGEKPYQC   73.5  
KLSHLQMHSRKHTGEKPYQ   68.5  
KLSHLQMHSRKHTGEKPY   73.1  
KLSHLQMHSRKHTGEKP   58.6  
KLSHLQMHSRKHTGEK   70.2  
KLSHLQMHSRKHTGE   71.5  
KLSHLQMHSRKHTG   71.2  
KLSHLQMHSRKHT   73.1  
KLSHLQMHSRKH   72.7  
KLSHLQMHSRK   32.5  
KLSHLQMHSR   7.3  
KLSHLQMHS   7.5  
LSHLQMHSRKHTGEKPYQC   63.9  
SHLQMHSRKHTGEKPYQC   32.4  
HLQMHSRKHTGEKPYQC   6.6  
LQMHSRKHTGEKPYQC   6.9  
QMHSRKHTGEKPYQC   4.9  
MHSRKHTGEKPYQC   6.4  
HSRKHTGEKPYQC   5.9  
SRKHTGEKPYQC   7.2  
RKHTGEKPYQC   3.5  
KHTGEKPYQC   7.8  
HTGEKPYQC   7.7  
LSHLQMHSRKH
 
72.1
 

WT1 peptide loaded

% cytotoxicity, at 10:1 E/T ratio
None   3.5  
KLSHLQMHSRKHTGEKPYQC   73.5  
KLSHLQMHSRKHTGEKPYQ   68.5  
KLSHLQMHSRKHTGEKPY   73.1  
KLSHLQMHSRKHTGEKP   58.6  
KLSHLQMHSRKHTGEK   70.2  
KLSHLQMHSRKHTGE   71.5  
KLSHLQMHSRKHTG   71.2  
KLSHLQMHSRKHT   73.1  
KLSHLQMHSRKH   72.7  
KLSHLQMHSRK   32.5  
KLSHLQMHSR   7.3  
KLSHLQMHS   7.5  
LSHLQMHSRKHTGEKPYQC   63.9  
SHLQMHSRKHTGEKPYQC   32.4  
HLQMHSRKHTGEKPYQC   6.6  
LQMHSRKHTGEKPYQC   6.9  
QMHSRKHTGEKPYQC   4.9  
MHSRKHTGEKPYQC   6.4  
HSRKHTGEKPYQC   5.9  
SRKHTGEKPYQC   7.2  
RKHTGEKPYQC   3.5  
KHTGEKPYQC   7.8  
HTGEKPYQC   7.7  
LSHLQMHSRKH
 
72.1
 

Cytotoxicity of NIK-1 against autologous LCL loaded and unloaded with various WT1 peptides. E/T ratio indicates effector-target ratio.

Because NIK-1 showed WT1 peptide-specific cytotoxicity, we addressed the question of whether NIK-1 can lyse leukemia cells. Because most leukemia cell lines are HLA class II negative, only one HLA-DP5-positive leukemia cell line expressing WT1 was available. As shown in Table 4, NIK-1 exerted strong cytotoxicity against HLA-DP5-positive WT1-expressing leukemia cell lines but not against HLA-DP5-negative leukemia cell lines or HLA-DP5-positive or HLA-DP5-negative lymphoma cell lines that are negative for WT1 expression. Similarly to the cytotoxicity against cell lines, HLA-DP5-positive but not HLA-DP5-negative freshly isolated leukemia cells were lysed efficiently by NIK-1. Cytotoxicity of leukemia cells mediated by NIK-1 appeared to be restricted by HLA-DP5, because only HLA-DP5-positive leukemia cells were lysed by NIK-1 and the cytotoxicity of leukemia cells mediated by NIK-1 was inhibited by adding anti-HLA-DP mAb (Table 5).

Table 4.

Direct recognition and lysis of leukemia cells by NIK-1: experiment 1



WT1 expression level

HLA-DP5 expression

% cytotoxicity, at E/T ratio
Target cells
20:1 ratio
10:1 ratio
5:1 ratio
Cell lines      
C2F8; leukemia   2.3 × 100  +   38.8   34.4   31.2  
MEG01; leukemia   8.4 × 10−1  −   3.6   1.8   1.2  
KAZZ; leukemia   1.1 × 100  −   2.1   2.7   0.9  
IZU; lymphoma   1.1 × 10−5  +   1.5   0.5   1.0  
IKE; lymphoma   8.6 × 10−5  +   0.7   1.8   0.5  
Daudi; lymphoma   3.8 × 10−5  −   2.0   0.3   0.4  
Freshly isolated leukemia cells      
From donor no. 1 (AML M1)   3.5 × 10−1  +   45.1   34.2   21.0  
From donor no. 2 (AML M2)   2.7 × 10−1  +   33.6   24.3   18.9  
From donor no. 3 (AML M2)   8.6 × 10−2  +   31.2   18.9   13.1  
From donor no. 4 (AML M1)   6.5 × 10−1  −   2.7   2.1   0.6  
From donor no. 5 (AML M2)   7.2 × 10−1  −   1.4   1.8   1.0  
From donor no. 6 (ALL L2)
 
5.8 × 10−1
 

 
0.0
 
0.3
 
0.0
 


WT1 expression level

HLA-DP5 expression

% cytotoxicity, at E/T ratio
Target cells
20:1 ratio
10:1 ratio
5:1 ratio
Cell lines      
C2F8; leukemia   2.3 × 100  +   38.8   34.4   31.2  
MEG01; leukemia   8.4 × 10−1  −   3.6   1.8   1.2  
KAZZ; leukemia   1.1 × 100  −   2.1   2.7   0.9  
IZU; lymphoma   1.1 × 10−5  +   1.5   0.5   1.0  
IKE; lymphoma   8.6 × 10−5  +   0.7   1.8   0.5  
Daudi; lymphoma   3.8 × 10−5  −   2.0   0.3   0.4  
Freshly isolated leukemia cells      
From donor no. 1 (AML M1)   3.5 × 10−1  +   45.1   34.2   21.0  
From donor no. 2 (AML M2)   2.7 × 10−1  +   33.6   24.3   18.9  
From donor no. 3 (AML M2)   8.6 × 10−2  +   31.2   18.9   13.1  
From donor no. 4 (AML M1)   6.5 × 10−1  −   2.7   2.1   0.6  
From donor no. 5 (AML M2)   7.2 × 10−1  −   1.4   1.8   1.0  
From donor no. 6 (ALL L2)
 
5.8 × 10−1
 

 
0.0
 
0.3
 
0.0
 

Cytotoxicity of NIK-1 against various cell lines and freshly isolated leukemia cells. E/T ratio indicates effector-target ratio.

Table 5.

Direct recognition and lysis of leukemia cells by NIK-1: experiment 2


Target cells

% cytotoxicity, at 10:1 E/T ratio
C2F8 cells  
No anti-HLA mAb   27.6  
With anti-HLA-DR mAb   28.0  
With anti-HLA-DQ mAb   30.3  
With anti-HLA-DP mAb   3.1  
Freshly isolated leukemia cells  
From donor no. 1 (AML M1)   
No anti-HLA mAb   45.1  
With anti-HLA-DR mAb   43.2  
With anti-HLA-DQ mAb   41.6  
With anti-HLA-DP mAb   5.6  
From donor no. 2 (AML M2)   
No anti-HLA mAb   33.6  
With anti-HLA-DR mAb   31.7  
With anti-HLA-DQ mAb   35.1  
With anti-HLA-DP mAb
 
5.6
 

Target cells

% cytotoxicity, at 10:1 E/T ratio
C2F8 cells  
No anti-HLA mAb   27.6  
With anti-HLA-DR mAb   28.0  
With anti-HLA-DQ mAb   30.3  
With anti-HLA-DP mAb   3.1  
Freshly isolated leukemia cells  
From donor no. 1 (AML M1)   
No anti-HLA mAb   45.1  
With anti-HLA-DR mAb   43.2  
With anti-HLA-DQ mAb   41.6  
With anti-HLA-DP mAb   5.6  
From donor no. 2 (AML M2)   
No anti-HLA mAb   33.6  
With anti-HLA-DR mAb   31.7  
With anti-HLA-DQ mAb   35.1  
With anti-HLA-DP mAb
 
5.6
 

Cytotoxicity of NIK-1 against leukemia cell line and freshly isolated leukemia cells in the presence or absence of anti-HLA-DR, anti-HLA-DQ, or anti-HLA-DP mAb.

E/T ratio indicates effector-target ratio.

To further confirm that the cytotoxicity of NIK-1 against leukemia cells was mediated by the specific recognition of endogenously processed WT1, we performed cold-target inhibition experiments. As shown in Table 6, the addition of WT1 peptide-loaded autologous LCL decreased the cytotoxicity of NIK-1 against leukemia cells, whereas the addition of peptide-unloaded autologous LCL had no effect on the cytotoxicity. These data strongly suggest that WT1 is naturally processed in leukemia cells and recognized by WT1-specific CD4+ CTLs in the context of HLA-DP5.

Table 6.

Direct recognition and lysis of leukemia cells by NIK-1: experiment 3



% cytotoxicity, at cold-hot target cell ratio
Hot target cells and cold target cells
0
5:1
10:1
20:1
C2F8 cells     
WT1 peptide-loaded autologous LCL   31.3   22.5   14.3   9.6  
Peptide-unloaded autologous LCL   31.3   30.5   29.7   28.1  
Freshly isolated leukemia cells from donor no. 1 (AML M1)     
WT1 peptide-loaded autologous LCL   42.1   26.4   15.2   8.8  
Peptide-unloaded autologous LCL
 
42.1
 
41.3
 
39.3
 
40.2
 


% cytotoxicity, at cold-hot target cell ratio
Hot target cells and cold target cells
0
5:1
10:1
20:1
C2F8 cells     
WT1 peptide-loaded autologous LCL   31.3   22.5   14.3   9.6  
Peptide-unloaded autologous LCL   31.3   30.5   29.7   28.1  
Freshly isolated leukemia cells from donor no. 1 (AML M1)     
WT1 peptide-loaded autologous LCL   42.1   26.4   15.2   8.8  
Peptide-unloaded autologous LCL
 
42.1
 
41.3
 
39.3
 
40.2
 

Cytotoxicity of NIK-1 against leukemia cells in the presence or absence of WT1 peptide-loaded autologous LCL or peptide-unloaded autologous LCL at an effector-hot target cell ratio of 10:1.

We then addressed the issue of whether NIK-1 recognizes WT1 peptide expressed on normal bone marrow progenitor cells and suppresses their growth. As shown in Table 7, after coculture with NIK-1 in the absence of WT1 peptide, the numbers of granulocyte-macrophage colony-forming units (CFU-GMs) and erythroid burst-forming units (BFU-Es) generated from bone marrow cells of 2 HLA-DP5-positive individuals were almost the same as those generated from bone marrow cells cultured alone. However, the numbers of CFU-GMs and BFU-Es decreased significantly when HLA-DP5-positive bone marrow cells were pretreated with WT1 peptide and then cocultured with NIK-1. As expected, NIK-1 had no effect on colony formation by HLA-DP5-negative bone marrow cells that had been pretreated with WT1 peptide or left untreated.

Table 7.

Direct recognition and lysis of leukemia cells by NIK-1: experiment 4



Colony formation
Donor
CFU-GM
BFU-E
HLA-DP5-positive donor no. 1   
Without NIK-1   56 ± 5   86 ± 8  
With NIK-1   59 ± 7   93 ± 10  
With NIK-1 and WT1 peptide   29 ± 3   37 ± 7  
HLA-DP5-positive donor no. 2   
Without NIK-1   136 ± 14   167 ± 24  
With NIK-1   138 ± 8   170 ± 23  
With NIK-1 and WT1 peptide   66 ± 14   62 ± 13  
HLA-DP5-negative donor no. 3   
Without NIK-1   86 ± 15   96 ± 18  
With NIK-1   88 ± 7   100 ± 10  
With NIK-1 and WT1 peptide
 
85 ± 15
 
103 ± 8
 


Colony formation
Donor
CFU-GM
BFU-E
HLA-DP5-positive donor no. 1   
Without NIK-1   56 ± 5   86 ± 8  
With NIK-1   59 ± 7   93 ± 10  
With NIK-1 and WT1 peptide   29 ± 3   37 ± 7  
HLA-DP5-positive donor no. 2   
Without NIK-1   136 ± 14   167 ± 24  
With NIK-1   138 ± 8   170 ± 23  
With NIK-1 and WT1 peptide   66 ± 14   62 ± 13  
HLA-DP5-negative donor no. 3   
Without NIK-1   86 ± 15   96 ± 18  
With NIK-1   88 ± 7   100 ± 10  
With NIK-1 and WT1 peptide
 
85 ± 15
 
103 ± 8
 

The colony formation by normal bone marrow cells cocultured with or without NIK-1 cells in the presence or absence of WT1 peptide.

In the present study, we demonstrated for the first time the generation of WT1-specific CD4+ T lymphocytes that can recognize directly leukemia cells in an HLA class II-restricted manner. It is well known that induction of the CD8+ CTL response requires cognate CD4+ T-lymphocyte help.17  CD4+ T lymphocytes recognize major histocompatibility complex (MHC) class II-binding peptides on antigen-presenting cells, such as dendritic cells (DCs), and their interaction may result not only in activation and priming of CD4+ T lymphocytes but also in activation of the DCs themselves.18  Consequent to this mutual activation, DCs prime and activate CD8+ CTLs specific for tumor-associated antigens. On the basis of this scenario, it is expected that WT1-specific CD4+ T lymphocytes may be effective for efficient induction of WT1-specific CD8+ CTLs in vivo.

The other interesting finding of this study is that WT1 peptide-specific CD4+ T lymphocytes exerted strong cytotoxicity against WT1-expressing leukemia cells in an HLA class II-restricted manner. In general, endogenous antigens are degraded in the cytoplasm to oligopeptides and bind to newly synthesized MHC class I molecules. On the other hand, exogenous antigens are processed into peptides capable of binding to MHC class II molecules. However, it has recently been shown that the MHC class II pathway can process and present endogenous antigens as well as exogenous antigens. For example, virus-infected cells are recognized by CD4+ T lymphocytes in a viral antigen-specific and MHC class II-restricted manner in vitro and in vivo.19,20  It has also been reported that tumor cells transfected with syngeneic MHC class II genes could present endogenously synthesized tumor-associated protein-derived peptides in the context of MHC class II molecules to CD4+ T lymphocytes.21  Taken together with previous data, our present findings strongly suggest that leukemia cells can process and present endogenously synthesized WT1 protein to CD4+ T lymphocytes in the context of HLA class II molecules.

In summary, we have demonstrated WT1-specific CD4+ T lymphocytes capable of producing Th1 cytokines and exerting direct cytotoxicity against leukemia cells in an HLA class II-restricted manner. Because most types of leukemic cells are positive for HLA class II expression,22  WT1-specific CD4+ CTLs may play an important role in the antileukemia response through cytotoxic activity as well as helper function for CD8+ CTL induction. On the basis of this concept, we are planning a clinical trial of WT1 peptide vaccination using a combination of peptides derived from epitopes recognized by CD4+ T lymphocytes as well as CD8+ T lymphocytes.

Prepublished online as Blood First Edition Paper, April 21, 2005; DOI 10.1182/blood-2005-01-0413.

Supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a Grant-in-Aid for Cancer Research (15-17) from the Ministry of Health, Labor and Welfare.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

We are grateful for the skilled technical assistance of Ms Yuka Sato. We thank Dr Hiroo Saji for HLA typing.

1
Xue S, Gao L, Gillmore R, et al. WT1-targeted immunotherapy of leukaemia.
Blood Cells Mol Dis.
2004
;
33
:
288
-290.
2
Rosenfeld C, Cheever MA, Gaiger A. WT1 in acute leukemia, chronic myelogenous leukemia and myelodysplastic syndrome: therapeutic potential of WT1 targeted therapies.
Leukemia.
2003
;
17
:
1301
-1312.
3
Sugiyama H. Cancer immunotherapy targeting WT1 protein.
Int J Hematol.
2002
;
76
:
127
-132.
4
Yasukawa M. Immunotherapy for leukemia targeting the Wilms' tumor gene.
Leuk Lymphoma.
2001
;
42
:
267
-273.
5
Ohminami H, Yasukawa M, Fujita S. HLA class I-restricted lysis of leukemia cells by a CD8+ cytotoxic T-lymphocyte clone specific for WT1 peptide.
Blood.
2000
;
95
:
286
-293.
6
Oka Y, Elisseeva OA, Tsuboi A, et al. Human cytotoxic T-lymphocyte responses specific for peptides of the wild-type Wilms' tumor gene (WT1) product.
Immunogenetics.
2000
;
51
:
99
-107.
7
Gao L, Bellantuono I, Elsasser A, et al. Selective elimination of leukemic CD34+ progenitor cells by cytotoxic T lymphocytes specific for WT1.
Blood.
2000
;
95
:
2198
-2203.
8
Gaiger A, Reese V, Disis ML, Cheever MA. Immunity to WT1 in the animal model and in patients with acute myeloid leukemia.
Blood.
2000
;
96
:
1480
-1489.
9
Doubrovina ES, Doubrovin MM, Lee S, et al. In vitro stimulation with WT1 peptide-loaded Epstein-Barr virus-positive B cells elicits high frequencies of WT1 peptide-specific T cells with in vitro and in vivo tumoricidal activity.
Clin Cancer Res.
2004
;
10
:
7207
-7219.
10
Oka Y, Tsuboi A, Taguchi T, et al. Induction of WT1 (Wilms' tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression.
Proc Natl Acad Sci U S A.
2004
;
101
:
13885
-13890.
11
Mailander V, Scheibenbogen C, Thiel E, Letsch A, Blau IW, Keilholz U. Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity.
Leukemia.
2004
;
18
:
165
-166.
12
Wang RF. The role of MHC class II-restricted tumor antigens and CD4+ T cells in antitumor immunity.
Trends Immunol.
2001
;
22
:
269
-276.
13
Yasukawa M, Ohminami H, Kaneko S, et al. CD4+ cytotoxic T-cell clones specific for bcr-abl b3a2 fusion peptide augment colony formation by chronic myelogenous leukemia cells in a b3a2-specific and HLA-DR-restricted manner.
Blood.
1998
;
92
:
3355
-3361.
14
Yasukawa M, Inatsuki A, Kobayashi Y. Helper activity in antigen-specific antibody production mediated by CD4+ human cytotoxic T cell clones directed against herpes simplex virus.
J Immunol.
1988
;
140
:
3419
-3425.
15
Makita M, Hiraki A, Azuma T, et al. Antilung cancer effect of WT1-specific cytotoxic T lymphocytes.
Clin Cancer Res.
2002
;
8
:
2626
-2631.
16
Inoue K, Sugiyama H, Ogawa H, et al. WT1 as a new prognostic factor and a new marker for the detection of minimal residual disease in acute leukemia.
Blood.
1994
;
84
:
3071
-3079.
17
Bennett SR, Carbone FR, Karamalis F, Miller JF, Heath WR. Induction of a CD8+ cytotoxic T lymphocyte response by cross-priming requires cognate CD4+ T cell help.
J Exp Med.
1997
;
186
:
65
-70.
18
Ridge JP, Di Rosa F, Matzinger P. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell.
Nature.
1998
;
393
:
474
-478.
19
Yasukawa M, Zarling JM. Human cytotoxic T cell clones directed against herpes simplex virus-infected cells, I: lysis restricted by HLA class II MB and DR antigens.
J Immunol.
1984
;
133
:
422
-427.
20
Jellison ER, Kim SK, Welsh RM. MHC class II-restricted killing in vivo during viral infection.
J Immunol.
2005
;
174
:
614
-618.
21
Armstrong TD, Clements VK, Ostrand-Rosenberg S. MHC class II-transfected tumor cells directly present antigen to tumor-specific CD4+ T lymphocytes.
J Immunol.
1998
;
160
:
661
-666.
22
Wetzler M, McElwain BK, Stewart CC, et al. HLA-DR antigen-negative acute myeloid leukemia.
Leukemia.
2003
;
17
:
707
-715.