Direct recognition and lysis of leukemia cells by WT1-specific CD4⁺ T lymphocytes in an HLA class II-restricted manner

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.

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).

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).

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.

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.

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.

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