A novel pyroptosis-related gene signature exhibits distinct immune cells infiltration landscape in Wilms' tumor.

BACKGROUND: Wilms' tumor (WT) is the most common renal tumor in childhood. Pyroptosis, a type of inflammation-characterized and immune-related programmed cell death, has been extensively studied in multiple tumors. In the current study, we aim to construct a pyroptosis-related gene signature for predicting the prognosis of Wilms' tumor. METHODS: We acquired RNA-seq data from TARGET kidney tumor projects for constructing a gene signature, and snRNA-seq data from GEO database for validating signature-constructing genes. Pyroptosis-related genes (PRGs) were collected from three online databases. We constructed the gene signature by Lasso Cox regression and then established a nomogram. Underlying mechanisms by which gene signature is related to overall survival states of patients were explored by immune cell infiltration analysis, differential expression analysis, and functional enrichment analysis. RESULTS: A pyroptosis-related gene signature was constructed with 14 PRGs, which has a moderate to high predicting capacity with 1-, 3-, and 5-year area under the curve (AUC) values of 0.78, 0.80, and 0.83, respectively. A prognosis-predicting nomogram was established by gender, stage, and risk score. Tumor-infiltrating immune cells were quantified by seven algorithms, and the expression of CD8( +) T cells, B cells, Th2 cells, dendritic cells, and type 2 macrophages are positively or negatively correlated with risk score. Two single nuclear RNA-seq samples of different histology were harnessed for validation. The distribution of signature genes was identified in various cell types. CONCLUSIONS: We have established a pyroptosis-related 14-gene signature in WT. Moreover, the inherent roles of immune cells (CD8( +) T cells, B cells, Th2 cells, dendritic cells, and type 2 macrophages), functions of differentially expressed genes (tissue/organ development and intercellular communication), and status of signaling pathways (proteoglycans in cancer, signaling pathways regulating pluripotent of stem cells, and Wnt signaling pathway) have been elucidated, which might be employed as therapeutic targets in the future.

EV PD-L1 Contributes to Immunosuppressive CD8+ T Cells in Peripheral Blood of Pediatric Wilms Tumor.

Wilms tumor (WT) is the most common renal cancer and the most prevalent abdominal cancer in children. Children with recurrent or progressive forms of WT could benefit from novel immune-targeted approaches. While the immune status of these patients, especially the immunosuppression of peripheral T cells, was rarely reported. The present study enrolled a consecutive series of 14 Chinese WT children and 14 age- and gender-matched healthy controls. We demonstrated that plasma extracellular vesicular (EV) PD-L1 levels significantly increased in WT patients than in healthy controls. EV PD-L1 significantly inhibited the activation of human CD8+ T cells by down-regulating the cell surface CD69 expression and the intracellular IFNγ and TNFα production in vitro. In peripheral CD8+ T cells of WT patients, the intracellular IFNγ and TNFα production significantly decreased than healthy controls. The level of plasma EV PD-L1 significantly correlated with the intracellular TNFα production in peripheral CD8+ T cells of WT patients. In conclusion, the significantly increased plasma EV PD-L1 in WT patients contributed to the immunosuppression of peripheral CD8+ T cells. Monitoring the level of plasma EV PD-L1 will be helpful for the selection of immune-targeted therapies for WT patients.

Gene expression-based immune infiltration analyses of renal cancer and their associations with survival outcome.

BACKGROUND: Renal cancer is a common malignant tumor with an increasing incidence rate. METHODS: In this study, based on the gene expression profiles, we analyzed the compositions of tumor-infiltrating immune cells (TIICs) in renal cancer and paracancerous samples using CIBERSORT. The proportions of 22 TIICs subsets in 122 paired renal carcinoma and paracancerous samples, and 224 Wilms tumor (WT) samples varied between intragroup and intergroup. RESULTS: After analyzed the difference of TIICs composition between renal cancer and paired paracancerous samples, we found that M0 macrophages and CD8 T cells were significantly elevated, while naive B cells were significantly decreased in renal cancer samples compared with paracancerous samples. Survival analysis showed that high overall TIICs proportion, the low proportion of resting mast cells and the high proportion of activated memory CD4 T cells were associated with poor prognosis of renal cancer patients. In addition, 3 clusters were identified by hierarchical clustering analysis, and they presented a distinct prognosis. Cluster 1 had superior survival outcomes, while cluster 2 had an inferior survival outcome. CONCLUSIONS: Our study indicated that overall TIICs proportion, certain TIICs subset proportion, including resting mast cells and activated memory CD4 T cells, and distinct cluster patterns were associated with the prognosis of renal cancer, which was significant for the clinical surveillance and treatment of renal cancer.

Immune checkpoint inhibitors in Wilms' tumor and Neuroblastoma: What now?

BACKGROUND: Therapeutic activation of tumor-infiltrating lymphocytes using monoclonal antibodies targeting PD1 or PD-L1 (immune checkpoint inhibitors-ICIs) has revolutionized treatment of specific solid tumors in adult cancer patients, and much hope has been placed on a similar effect in relapsed or refractory solid pediatric tumors. Recent clinical trials have disappointingly shown an almost nonexistent response rate, while case reports have demonstrated that some pediatric patients do achieve durable responses when treated with this type of drug. AIM: To elucidate this paradox, we mapped the landscape of expressed neoantigens as well as the levels of immune cell infiltration in the two most common extracranial solid pediatric tumors: Wilms tumor and neuroblastoma using state-of-the-art in silico analysis of a large cohort of patients with these tumors. METHODS: By integration of whole exome sequencing and RNA-sequencing, we mapped the landscape of neoantigens in the TARGET cohorts for these diagnoses and correlated these findings with known genetic prognostic markers. RESULTS: Our analysis shows that these tumors typically have much lower levels of expressed neoantigens than commonly seen in adult cancers, but we also identify subgroups with significantly higher levels of neoantigens. For neuroblastomas, the cases with higher levels of neoantigens were confined to the group without MYCN-amplification and for Wilms tumor restricted to the TP53-mutated cases. Furthermore, we demonstrate that neuroblastomas have an unexpectedly high level of CD8+ tumor-infiltrating lymphocytes, even when compared to adult tumor types where ICI is an approved treatment. CONCLUSION: These results could be important to consider when designing future clinical trials of ICI treatment in pediatric patients with relapsed or refractory solid tumors.

Impact of mature dendritic cells pulsed with a novel WT1 helper peptide on the induction of HLA­A2­restricted WT1­reactive CD8+ T cells.

The proliferation and activation of CD4+ T helper 1 (Th1) cells and CD8+ cytotoxic T lymphocytes (CTLs) that produce interferon­Î³ (IFN­Î³) is an essential action of effective cancer vaccines. Recently, a novel Wilms' tumor 1 (WT1) helper peptide (WT1 HP34­51; amino acid sequence, WAPVLDFAPPGASAYGSL) applicable for various human leukocyte antigen (HLA) subtypes (HLA­DR, HLA­DP and HLA­DQ) was reported to increase peptide immunogenicity; however, the function of WT1 HP34­51 remains unclear. In the present study, mature dendritic cells (mDCs) pulsed with WT1 HP34­51 (mDC/WT1 HP34­51) activated not only WT1­specific CD4+ T cells but also CD8+ T cells that produced IFN­Î³ following stimulation with immature dendritic cells (imDCs) pulsed with WT1 killer peptide (imDC/WT1 KP37­45) in an HLA­A*02:01­ or HLA­A*02:06­restricted manner. Furthermore, the activated WT1­reactive CD4+ Th1 cells were predominantly effector memory (EM) T cells. In 5 of 12 (41.7%) patients with cancer carrying the HLA­A*02:01 or HLA­A*02:06 allele, WT1­reactive CD8+ T cells stimulated with mDC/WT1 HP34­51 enhanced their levels of WT1 KP37­45­specific IFN­Î³ production, with an increase >10%. Simultaneous activation of CD4+ and CD8+ T cells occurred more often when stimulation with mDC/WT1 HP34­51 was combined with imDC/WT1 KP37­45 restimulation. These results indicated that the novel mDC/WT1 HP34­51 combination induced responses by WT1­specific EM CD4+ Th1 cells and HLA­A*02:01­ or HLA­A*02:06­restricted CD8+ CTLs, suggesting its potential as a WT1­targeting cancer vaccine.

The Antitumor Activity of TCR-Mimic Antibody-Drug Conjugates (TCRm-ADCs) Targeting the Intracellular Wilms Tumor 1 (WT1) Oncoprotein.

Wilms tumor 1 (WT1) oncoprotein is an intracellular oncogenic transcription factor which is barely expressed in normal adult tissues but over expressed in a variety of leukemias and solid cancers. WT1-derived HLA-A*02:01 T cell epitope, RMFPNAPYL (RMF), is a validated target for T cell-based immunotherapy. We generated two T cell receptor mimic antibody-drug conjugates (TCRm-ADCs), ESK-MMAE, and Q2L-MMAE, against WT1 RMF/HLA-A*02:01 complex with distinct affinities, which mediate specific antitumor activity. Although ESK-MMAE showed higher tumor growth inhibition ratio in vivo, the efficacy of them was not so promising, which might be due to low expression of peptide/HLA targets. Therefore, we explored a bispecific TCRm-ADC that exerted more potent tumor cytotoxicity compared with TCRm-ADCs. Hence, our findings validate the feasibility of the presenting intracellular peptides as the targets of ADCs, which broadens the antigen selection range of antibody-based drugs and provides new strategies for precision medicine in tumor therapy.

Immune expression in children with Wilms tumor: a pilot study.

BACKGROUND: Given improvements in multimodality therapy, survival among children with Wilms tumor (WT) exceeds 90%. However, 15% of children with favorable histology and 50% of children with anaplastic WT experience recurrence or progression. Of patients with advanced disease, only 50% survive to adulthood. In adult malignancies (including renal tumors), patient survival has improved with the advent of immunotherapy. However, little is known about the immune microenvironment of WT, making the potential role of immunotherapy unclear. OBJECTIVE: The objective of the study is to perform an exploratory, descriptive analysis of the immune milieu in WT. STUDY DESIGN: Between 2016 and 2017, all pediatric patients with WT, some of whom received neoadjuvant chemotherapy, underwent ex vivo wedge biopsy at the time of nephrectomy. The fresh tumor tissue and peripheral blood samples were analyzed for infiltrating immune infiltrate and effector cells using flow cytometry. Immunohistochemistry was performed for CD4, CD8, and PD-L1 expression. Matched blood samples were obtained for each patient, and circulating immune cells were analyzed by flow cytometry. RESULTS: A total of six patients were enrolled. One patient with neuroblastoma was excluded. The remaining five patients included the following: two with unilateral WT (resected before chemotherapy), two with bilateral WT (resected after neoadjuvant chemotherapy), and one with Denys-Drash syndrome, end-stage renal disease, and history of WT in the contralateral kidney. Immune analysis showed that WT were infiltrated by immune cells regardless of chemotherapy status. CD8 and CD4 T cells were present in the tumor tissue and exhibited an activated phenotype. Elevated levels of natural killer (NK) cells were observed in the tumors (Figure). Immune checkpoint PD-L1 was also found expressed in one of the tumors stained. DISCUSSION: In this pilot study, it was found that WTs were infiltrated by immune cells (CD45+) both before and after chemotherapy. Elevated levels of NK cells infiltrating the tumor specimens, which were quantitatively increased compared with levels of NK cells circulating in the blood, were noted. T cells, particularly CD4+ and CD8+ T cells, were present in tumor specimens. Tumor-infiltrating CD4 and CD8 T cells displayed an activated phenotype as defined by increased expression of human leukocyte antigen-DR isotype (HLA-DR), programmed cell death protein 1 (PD1), and CD57. Together, these findings suggest that WT microenvironment is immune engaged and may be susceptible to immunotherapy similar to other malignancies. CONCLUSIONS: These pilot data suggest an immune-engaged tumor microenvironment is present within WT. This implies that WT may be susceptible to immunotherapy similar to adult renal tumors and other adult malignancies. Follow-up studies are currently underway.

Significance of Wilms' tumor 1 antigen as a cancer vaccine for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by a very poor prognosis, despite novel chemotherapeutic treatments. Moreover, the majority of PDA patients with complete surgical resection show recurrence within 5 years of resection. Therefore, new targeted cancer vaccines are urgently needed to extend PDA patient survival. The Wilms' tumor 1 (WT1) antigen was identified as an excellent antigen in a list of 75 tumor-associated antigens by a National Cancer Institute prioritization project based on several factors, such as therapeutic function. WT1-targeted cancer vaccines are not only efficient in the regression of PDA cells but also angiogenesis and immune suppressive cells, such as myeloid-derived suppressor cells (MDSCs), in the PDA microenvironment. Moreover, WT1 is increased in PDA cells; thus, WT1 may represent an effective therapeutic target for PDA, resulting in a survival benefit for PDA patients. A novel WT1 peptide with increased immunogenicity was developed and used in clinical trials to induce more successful clinical results. This review summarizes the clinical trials of PDA patients receiving WT1-targeted cancer vaccines.

Adaptive from Innate: Human IFN-γ+CD4+ T Cells Can Arise Directly from CXCL8-Producing Recent Thymic Emigrants in Babies and Adults.

We recently demonstrated that the major effector function of neonatal CD4+ T cells is to produce CXCL8, a prototypic cytokine of innate immune cells. In this article, we show that CXCL8 expression, prior to proliferation, is common in newly arising T cells (so-called "recent thymic emigrants") in adults, as well as in babies. This effector potential is acquired in the human thymus, prior to TCR signaling, but rather than describing end-stage differentiation, such cells, whether isolated from neonates or adults, can further differentiate into IFN-γ-producing CD4+ T cells. Thus, the temporal transition of host defense from innate to adaptive immunity is unexpectedly mirrored at the cellular level by the capacity of human innate-like CXCL8-producing CD4+ T cells to transition directly into Th1 cells.

Molecular cloning of canine Wilms' tumor 1 for immunohistochemical analysis in canine tissues.

Wilms' tumor 1 (WT1) expression has been investigated in various human cancers as a target molecule for cancer immunotherapy. However, few studies have focused on WT1 expression in dogs. Firstly, cDNA of canine WT1 (cWT1) was molecularly cloned from normal canine kidney. The cross-reactivity of the anti-human WT1 monoclonal antibody (6F-H2) with cWT1 was confirmed via Western blotting using cells overexpressing cWT1. Immunohistochemical staining revealed that cWT1 expression was detected in all canine lymphoma tissues and in some normal canine tissues, including the kidney and lymph node. cWT1 is a potential immunotherapy target against canine cancers.

Role of Cyclooxygenase-2 Pathway in Creating an Immunosuppressive Microenvironment and in Initiation and Progression of Wilms' Tumor.

Wilms' tumors (WT), which accountfor 6% of all childhood cancers, arise from dysregulated differentiation of nephrogenic progenitor cells from embryonic kidneys. Though there is an improvement in the prognosis of WT, still 10% of patients with WT die due to recurrence. Thus more effective treatment approaches are necessary. We previously characterized the inflammatory microenvironment in human WT and observed the robust expression of COX-2. The aim of this study was to extend our studies to analyze the role of COX-2 pathway components in WT progression using a mouse model of WT. Herein, COX-2 pathway components such as COX-2, HIF1-α, p-ERK1/2, and p-STAT3 were upregulated in mouse and human tumor tissues. In our RPPA analysis, COX-2 was up-regulated in M15 cells after Wt1 gene was knocked down. Flow cytometry analysis showed the increased infiltration of immune suppressive inflammatory cells such as pDC's and Treg cells in tumors. The chemotactic chemokines responsible for the infiltration of these cells were also induced in CCR5 and CXCR4 dependent manner respectively. The immunosuppressive cytokines IL-10, TGF-ß, and TNF-α were also up-regulated. Furthermore, more pronounced Th2 and Treg induced cytokine response was observed than Th1 response in tumors. Basing on all these evidences it is speculated that COX-2 pathway may be a beneficial target for the treatment of WT. It may be most effective as an adjuvant therapy together with other inhibitors. Thus, our current study provides a good rationale for initiating animal studies to confirm the efficacy of COX-2 inhibitors in decreasing tumor cell growth in vivo.

[Expression of regulatory T cells and natural killer T cells in peripheral blood of children with Wilms tumor].

OBJECTIVE: To study the changes and clinical significance of CD4+CD25+CD127low regulatory T cells (Treg) and CD3+CD16+CD56+ natural killer T cells (NKT) in peripheral blood of children with Wilms tumor. METHODS: Twenty-one children with Wilms tumor were enrolled as the case group, and twenty-one healthy children for physical examinations were enrolled as the control group. Flow cytometry was used to detect the levels of CD4+CD25+CD127low T cells and CD3+CD16+CD56+ T cells in peripheral blood of two groups. RESULTS: The level of Treg cells in peripheral blood of the case group was significantly lower than in the control group (p<0.05). The level of NKT cells in peripheral blood of the case group was significantly higher than in the control group (p<0.05). CONCLUSIONS: Treg cells and NKT cells play important roles in the occurrence and development of Wilms tumor. Treg cells and NKT cells may be useful indexes for evaluating immunological function in children with Wilms tumor.

Programmed Death-Ligand 1 on Antigen-presenting Cells Facilitates the Induction of Antigen-specific Cytotoxic T Lymphocytes: Application to Adoptive T-Cell Immunotherapy.

Programmed death-ligand 1 (PD-L1) binds to programmed death-1 (PD-1) on activated T cells and contributes to T-cell exhaustion. PD-L1 expressed on antigen-presenting cells (APCs) could be thought to inhibit the induction of Ag-specific cytotoxic T lymphocytes (CTLs) by transducing negative signal into T cells; however, the roles of PD-L1 on APCs have not yet been well examined. Therefore, we evaluated the roles of PD-L1 on APCs in the induction of Ag-specific CTLs. CD3 T cells isolated from cytomegalovirus (CMV)-seropositive healthy donors were stimulated with mature dendritic cells pulsed with CMV pp65-derived HLA-restricted peptides in the presence of anti-PD-L1 blocking antibody. Unexpectedly, PD-L1 blockade resulted in a less efficient induction of CMV-specific CTLs, suggesting that PD-L1 play a positive role in the induction of Ag-specific CTLs. For further evaluations and application to adoptive immunotherapy, we generated K562-based artificial APCs, which were retrovirally transduced with HLA class I molecules and various combinations of CD80/86 and PD-L1. K562/HLA+CD80/86+PD-L1 cells produced significantly higher induction of CMV-specific CTLs than K562/HLA or K562/HLA+CD80/86 cells without causing excessive differentiation or functional exhaustion of the induced CTLs, whereas PD-L1 itself did not have a stimulatory effect. Furthermore, only K562/HLA+CD80/86+PD-L1 cells pulsed with HLA-A*24:02-restricted Wilms tumor 1 (WT1) peptide clearly expanded WT1-specific CTLs from healthy donors. Our findings presumed that PD-L1 expressed on APCs along with CD80/86 enhanced the induction of Ag-specific CTLs probably depending on fine-tuning excessive stimulation of CD80/86, and that K562/HLA+CD80/86+PD-L1 cells has therapeutic potential as a novel type of artificial APCs for adoptive immunotherapy.

Secondary Hemophagocytic Lymphohistiocytosis in a Patient With Favorable Histology Wilms Tumor.

Secondary hemophagocytic lymphohistiocytosis (HLH) is most commonly associated with malignancy, infection, or an underlying autoimmune disorder. Malignancy-associated hemophagocytic syndrome is responsible for most secondary HLH cases, but it has not been well described in children. We present a case of a 4-year-old female with favorable histology of Wilms tumor who developed secondary HLH after unsuccessful resection of the tumor and initiation of chemotherapy.

Wilms tumor 1 (WT1) protein: Diagnostic utility in pediatric tumors.

Despite Wilms tumor 1 (WT1) protein was originally considered as a specific immunomarker of Wilms tumor, with the increasing use of immunohistochemistry, there is evidence that other tumors may share WT1 protein expression. This review focuses on the immunohistochemical profile of WT1 protein in the most common malignant tumors of children and adolescents. The variable expression and distribution patterns (nuclear vs cytoplasmic) in the different tumors, dependent on the antibodies used (anti-C or N-terminus WT1 protein), will be emphasized by providing explicative illustrations. Potential diagnostic pitfalls from unexpected WT1 protein expression in some tumors will be discussed in order to avoid diagnostic errors, especially when dealing with small biopsies.

Orthotopic Wilms tumor xenografts derived from cell lines reflect limited aspects of tumor morphology and clinical characteristics.

BACKGROUND: Wilms tumor (WT) is a pediatric tumor of the kidney, the treatment of which includes heavy chemotherapy. Affected children would likely benefit from more targeted therapies with limited side effects. Establishment of relevant orthotopic WT xenografts is important to better understand mechanisms of WT growth and for preclinical drug testing. PROCEDURE: Here we established and characterized orthotopic xenografts from WT cell lines WiT49, CCG-99-11, and WT-CLS1 to ascertain in what aspects each of them recapitulated WT histology, immunophenotype, invasion, and metastatic spread. RESULTS: WiT49 xenografts recapitulated near triphasic WTs with clear WT1 staining and anaplastic features, but with tumor restricted to the kidney. On the contrary both CCG-99-11 and WT-CLS1 xenografts conveyed metastatic disease. CCG-99-11 showed a blastemal phenotype whereas WT-CLS1 xenografts did not properly reflect any specific WT subtype. CONCLUSIONS: From the three tested cell lines, orthotopic WiT49 xenografts best reflect the triphasic pattern of classical WT.

Targeting the intracellular WT1 oncogene product with a therapeutic human antibody.

The Wilms tumor 1 (WT1) oncoprotein is an intracellular, oncogenic transcription factor that is overexpressed in a wide range of leukemias and solid cancers. RMFPNAPYL (RMF), a WT1-derived CD8+ T cell human leukocyte antigen (HLA)-A0201 epitope, is a validated target for T cell-based immunotherapy. Using phage display technology, we discovered a fully human "T cell receptor-like" monoclonal antibody (mAb), ESK1, specific for the WT1 RMF peptide/HLA-A0201 complex. ESK1 bound to several leukemia and solid tumor cell lines and primary leukemia cells, in a WT1- and HLA-A0201-restricted manner, with high avidity [dissociation constant (Kd)=0.1 nM]. ESK1 mediated antibody-dependent human effector cell cytotoxicity in vitro. Low doses of naked ESK1 antibody cleared established, disseminated, human acute lymphocytic leukemia and Philadelphia chromosome-positive leukemia in nonobese diabetic/severe combined immunodeficient γc-/- (NSG) mouse models. At therapeutic doses, no toxicity was seen in HLA-A0201 transgenic mice. ESK1 is a potential therapeutic agent for a wide range of cancers overexpressing the WT1 oncoprotein. This finding also provides preclinical validation for the strategy of developing therapeutic mAbs targeting intracellular oncogenic proteins.

[WT1-targeting cancer vaccine].

Wilms' tumor gene WT1 encodes a transcription factor and functions as an oncogene. WT1 gene product WT1 protein is a promising par-tumor-associated antigen. WT1 peptide-based immunotherapy has been performing for more than six hundred patients with leukemias and various types of solid tumors. This immunotherapy is safe and has clinical benefit especially for leukemia, glioblastoma multiforme, advanced pancreatic cancer, and ovarian cancer. As a new strategy for cancer treatment, it should be recommended to initiate immunotherapy that had a potential of eradication of cancer stem cells before surgery, chemo- and radio-therapy.

Simultaneous immunisation with a Wilms' tumour 1 epitope and its ubiquitin fusions results in enhanced cell mediated immunity and tumour rejection in C57BL/6 mice.

Protein fusion to ubiquitin results in its targeting to proteasome and processing through MHC class I pathway. We used this approach to induce cytotoxic T lymphocyte (CTL) response against a MHC class I epitope. Therefore, two known proteasome targeting systems, "ubiquitin fusion degradation" (UFD) and "N-end rule", were used to immunise C57BL/6 mice. Two plasmids encoding an epitope from Wilms' Tumour 1 (WT1-126), fused N-terminally to ubiquitin, were constructed. They were designated as "pUbVVPT" and "pUbGRPT", targeting the fused epitope to UFD and N-end pathways, respectively. A plasmid encoding WT1-126 without ubiquitin fusion (pPT) was also constructed as control. Three mice groups were immunised using these constructs (UGR, UVV and PT groups). Two other groups received mixed immunisations of pUbVVPT or pUbGRPT plus pPT plasmids (UVV+PT and UGR+PT). All mice received a WT1-126 peptide booster. Lymphoproliferative responses following stimulation with WT1-126 were observed in all immunisation groups, with mice receiving the mixture of plasmids eliciting the highest proliferation (UVV+PT>UGR+PT>PT). Moreover, In vivo cytotoxicity assay results revealed highest specific lysis of target cells in UVV+PT group. Tumour growth was decreased in all immunised groups, and was completely abrogated in UGR+PT group. In addition, T(H)1 type cytokines patterns were detected from all immunised groups and WT1-126-specific IFNγ producing lymphocytes were developed in them. These results suggest that the delivery of ubiquitin-fused epitopes along with epitopes alone can be used to optimise the effect of DNA vaccines on the induction of anti-tumour immunity.