Identification of a WT1 protein‐derived peptide,WT1187, as a HLA‐A*0206‐restricted,WT1‐specific CTL epitope

The Wilms' tumor gene WT1 is overexpressed in various kinds of hematopoietic malignancies as well as solid cancers, and this protein has been demonstrated to be an attractive target antigen for cancer immunotherapy. WT1‐specific CTL epitopes with a restriction of HLA‐A*2402 or HLA‐A*0201 have been already identified. In the present study it has been demonstrated that a 9‐mer WT1‐derived WT1₁₈₇ peptide, which had already been shown to elicit a WT1‐specific CTL response with a restriction of HLA‐A*0201, can also elicit a CTL response with a restriction of HLA‐A*0206. In all three different HLA‐A*0206⁺ healthy donors examined, WT1₁₈₇ peptide‐specific CTL could be generated from peripheral blood mononuclear cells, and the CTL showed cytotoxic activity that depended on dual expression of WT1 and HLA‐A*0206 molecules. The present study describes the first identification of a HLA‐A*0206‐restricted, WT1‐specific CTL epitope. The present results should help to broaden the application of WT1 peptide‐based immunotherapy from only HLA‐A*0201‐positive to HLA‐A*0206‐positive cancer patients as well.     

Third generation dendritic cell vaccines for tumor immunotherapy

This review summarizes our studies of the past several years on the development of third generation dendritic cell (DC) vaccines. These developments have implemented two major innovations in DC preparation: first, young DCs are prepared within 3 days and, second, the DCs are matured with the help of Toll-like receptor agonists, imbuing them with the capacity to produce bioactive IL-12 (p70). Based on phenotype, chemokine-directed migration, facility to process and present antigens, and stimulatory capacity to polarize Th1 responses in CD4⁺ T cells, induce antigen-specific CD8⁺ CTL and activate natural killer cells, these young mDCs display all the important properties needed for initiating good antitumor responses in a vaccine setting.     

A WT1 protein‐derived,naturally processed 16‐mer peptide,WT1332, is a promiscuous helper peptide for induction of WT1‐specific Th1‐type CD4+ T cells

The Wilms' tumor gene WT1 is overexpressed in various tumors, and the WT1 protein has been demonstrated to be an attractive target antigen for cancer immunotherapy. A WT1 protein‐derived 16‐mer peptide, WT1₃₃₂ (KRYFKLSHLQMHSRKH), which was naturally generated through processing in cells and could elicit Th1‐type CD4⁺ helper T cell responses with an HLA‐DRB1*0405‐restriction has previously been identified by us. In the present study, it has been demonstrated that WT1₃₃₂ can induce WT1₃₃₂‐specific CD4⁺ T cell responses with the restriction of not only HLA‐DRB1*0405 but also HLA‐DRB1*1501, ‐DRB1*1502, or ‐DPB1*0901. These HLA class II‐restricted WT1₃₃₂‐specific CD4⁺ T cell lines produced IFN‐γ but neither IL‐4 nor IL‐10 with WT1₃₃₂ stimulation, thus showing a Th1‐type cytokine profile. Furthermore, HLA‐DRB1*1501 or ‐DRB1*1502‐restricted WT1₃₃₂‐specific CD4⁺ T cell lines responded to WT1‐expressing transformed cells in an HLA‐DRB1‐restricted manner, which is consistent with our previous finding that WT1₃₃₂ is a naturally processed peptide. These results indicate that the natural peptide, WT1₃₃₂, is a promiscuous WT1‐specific helper epitope. WT1₃₃₂ is expected to apply to cancer patients with various types of HLA class II as a WT1‐specific helper peptide in combination with HLA class I‐restricted WT1 peptides.     

The Wilms' tumor gene WT1 is a common marker of progenitor cells in fetal liver

It is well known that the Wilms' tumor gene WT1 plays an important role in cell proliferation and differentiation, and in organ development. In this study, to examine the role of the WT1 gene in lineage determination, fetal liver cells from LacZ-transgenic mice, in which WT1 expression was marked by the expression of the LacZ gene driven by WT1 promoter, were FACS-sorted according to LacZ expression of high (LacZ(++)) or undetectable (LacZ(-)) levels, which paralleled endogenous WT1 expression levels. LacZ(++) fetal liver cells were enriched by hepatocyte and endothelial progenitor cells. These results indicated that WT1 expression is a common marker of both hepatocyte and endothelial progenitors. These results also implied a role of the WT1 gene in lineage determination.     

WT1 peptide vaccination combined with BCG-CWS is more efficient for tumor eradication than WT1 peptide vaccination alone

A Wilms tumor gene WT1 is expressed at high levels not only in most types of leukemia but also in various types of solid tumors, including lung and breast cancer. WT1 protein has been reported to serve as a target antigen for tumor-specific immunotherapy both in vitro in human systems and in vivo in murine models. We have shown that mice immunized with WT1 peptide or WT1 cDNA could reject a challenge from WT1-expressing tumor cells (a prophylactic model). However, it was not examined whether WT1 peptide vaccination had the potency to reject tumor cells in a therapeutic setting. In the present study, we demonstrated for the first time that WT1 peptide vaccination combined with Mycobacterium bovis bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) was more effective for eradication of WT1-expressing tumor cells that had been implanted into mice before vaccination (a therapeutic model) compared with WT1 peptide vaccination alone. An intradermal injection of BCG-CWS into mice, followed by that of WT1 peptide at the same site on the next day, generated WT1-specific cytotoxic T lymphocytes (CTLs) and led to rejection of WT1-expressing leukemia or lung cancer cells. These results showed that BCG-CWS, which was well known to enhance innate immunity, could enhance WT1-specific immune responses (acquired immunity) in combination with WT1 peptide vaccination. Therefore, WT1 peptide vaccination combined with BCG-CWS may be applied to cancer immunotherapy in clinical settings.H. Nakajima and K. Kawasaki contributed equally to this study.     

Functional assays of HLA A2-restricted epitope variant of latent membrane protein 1 (LMP-1) of Epstein-Barr virus in nasopharyngeal carcinoma of Southern China and Taiwan

Summary Human leukocyte antigen (HLA) A2 was consistently associated with increased risk for nasopharyngeal carcinoma (NPC) in Chinese populations. Previously we have reported that an Epstein-Barr virus (EBV) strain carrying an HLA A2-restricted epitope variant of LMP-1 is prevalent in NPC in southern China and Taiwan (Lin et al., J. Gen. Virol. 85: 2023-2034, 2004). The variant has mutation selectively involved one of the two anchor residues in position 2 (125 L→F) and an additional mutation in position 5 (129 M→I). Functional assays of the epitope variant were carried out in the present work. The stabilization assay on T2 cells indicated that the variant peptide YFL (YFLEILWRL) prevalent in NPC binds to HLA A2 molecules less efficiently than the prototype peptide YLL (YLLEMLWRL). A dose-dependent binding of the HLA A2 molecules with added peptides was observed. In ex vivo cytotoxic T lymphocyte (CTL) assays with CD8-enriched effectors from A2-positive donors revealed that the YLL-specific CTL was able to lyse EBV-infected B cells expressing HLA A2, whereas the CTL recognition was abrogated with the peptide YFL. Cytokine (IFN-γ) responses, measured both by intracytoplasmic staining and ELISPOT assays after peptide stimulation, also indicated that the variant epitope peptide failed to give an IFN-γ response. The IFN-γ response was almost entirely restricted to those tetramer-positive cells. These results show that EBV isolates from NPC of southern China and Taiwan is dominated by an HLA A2-restricted 'epitope-loss variants' of LMP-1, which would allow the virus to resist immune recognition and may in part contribute to the prevalence of NPC in these populations.     

Downregulation of Wilms' tumor 1 protein inhibits breast cancer proliferation

High levels of Wilms' Tumor 1 (WT1) mRNA have been correlated with poor prognosis in breast cancer patients. However, the function of WT1 protein in breast cancer is not known. We observed that the levels of WT1 protein correlated with the proliferation of breast cancer cells. When the proliferation of breast cancer cells was stimulated by 17beta-estradiol, WT1 protein expression increased. But when the proliferation of breast cancer cells was inhibited by tamoxifen or all-trans retinoic acid (ATRA), WT1 protein expression decreased. We hypothesize that WT1 protein plays a role in regulating breast cancer cell proliferation. Using liposome-incorporated WT1 antisense oligodeoxynucleotides, we found that downregulation of WT1 protein expression led to breast cancer growth inhibition and reduced cyclin D1 protein levels. These results indicate that WT1 protein contributes to breast cancer progression by promoting breast cancer cell proliferation.     

The development of Wilms tumor: From WT1 and microRNA to animal models

Wilms tumor recapitulates the development of the kidney and represents a unique opportunity to understand the relationship between normal and tumor development. This has been illustrated by the findings that mutations of Wnt/β-catenin pathway-related WT1, β-catenin, and WTX together account for about one-third of Wilms tumor cases. While intense efforts are being made to explore the genetic basis of the other two-thirds of tumor cases, it is worth noting that, epigenetic changes, particularly the loss of imprinting of the DNA region encoding the major fetal growth factor IGF₂, which results in its biallelic over-expression, are closely associated with the development of many Wilms tumors. Recent investigations also revealed that mutations of Drosha and Dicer, the RNases required for miRNA generation, and Dis3L2, the 3′–5′ exonuclease that normally degrades miRNAs and mRNAs, could cause predisposition to Wilms tumors, demonstrating that miRNA can play a pivotal role in Wilms tumor development. Interestingly, Lin28, a direct target of miRNA let-7 and potent regulator of stem cell self-renewal and differentiation, is significantly elevated in some Wilms tumors, and enforced expression of Lin28 during kidney development could induce Wilms tumor. With the success in establishing mice nephroblastoma models through over-expressing IGF₂ and deleting WT1, and advances in understanding the ENU-induced rat model, we are now able to explore the molecular and cellular mechanisms induced by these genetic, epigenetic, and miRNA alterations in animal models to understand the development of Wilms tumor. These animal models may also serve as valuable systems to assess new treatment targets and strategies for Wilms tumor.     

Potent CTL induction by a whole cell pertussis vaccine in anti-tumor peptide immunotherapy

Promising yet limited clinical responses have been reported for peptide based immunotherapy against tumors. In order to induce more potent cytolytic CD8 T cell responses, we investigated the use of Bordetella pertussis vaccine as an adjuvant for peptide immunization. A whole cell (Wc) vaccine has been known to induce a Th1 biased immune response while an acellular (Ac) vaccine tends to induce that of the Th2 type. Natural infection by B. pertussis helps to maintain a robust Th1 memory in the host population. To examine the adjuvant activity of the pertussis vaccine, we immunized mice with an ovalbumin peptide as a model tumor antigen, and monitored the development of anti-tumor activities. The addition of either the Ac or the Wc vaccine helped expand the specific CD8 T cells. However, there was a marked difference in the induced cytolytic activity where the Wc vaccine was superior to the Ac. The Wc vaccine was also more effective in inducing in vivo tumor rejection. The adjuvant activity was not only effective against ovalbumin, but was also evident when an endogenous tumor antigen, Wilms' tumor 1 gene product, was targeted. These results indicate that, although the Wc vaccine does not share the same antigen specificity with tumor cells, it can aid in the development of highly cytolytic CD8 T cells as an adjuvant at the site of peptide immunization.     

Use of multicellular tumor spheroids to dissect endothelial cell-tumor cell interactions: a role for T-cadherin in tumor angiogenesis

This study addresses establishment of an "in vitro" melanoma angiogenesis model using multicellular tumor spheroids (MCTS) of differentiated (HBL) or undifferentiated (NA8) melanoma cell lines. DNA microarray assay and qRT-PCR indicated upregulation of pro-angiogenic factors IL-8, VEGF, Ephrin A1 and ANGPTL4 in NA8-MCTSs (vs. monolayers) whereas these were absent in MCTS and monolayer cultures of HBL. Upon co-culture with endothelial cell line HMEC-1 NA8-MCTS attract, whereas HBL-MCTS repulse, HMEC-1. Overexpression of T-cadherin in HMEC-1 leads to their increased invasion and network formation within NA8-MCTS. Given an appropriate angiogenic tumor microenvironment, T-cadherin upregulation on endothelial cells may potentiate intratumoral angiogenesis.