A clear correlation between WT1-specific Th response and clinical response in WT1 CTL epitope vaccination.

Clinical studies of WT1-targeted cancer vaccine are being performed. However, WT1-specific Th response in cancer patients remains unclear. Using quantitative real-time RT-PCR, we investigated IFN-gamma and IL-10 mRNA expression from Th cells by stimulation with helper peptide WT1(332). Seventeen patients, of whom 10 had achieved stable disease and the remaining 7 had progressive disease, were weekly vaccinated with WT1 CTL epitope (modified WT1(235)) and examined for WT1(332)-specific Th response. A clear correlation between WT1(332)-specific Th response and clinical response was observed at 4 weeks post-vaccination. In patients who responded, a clear inverse correlation between IL-10-type and IFN-gamma-type WT1(332)-specific Th response was detected at pre- and 4 weeks post-vaccination, and the shift of the Th response from IL-10-type dominancy at early phase to IFN-gamma-type dominancy at late phase was observed. From this study we concluded that occurrence of WT1(332)-specific Th response could predict good clinical response of WT1 CTL epitope vaccination.

High frequencies of less differentiated and more proliferative WT1-specific CD8+ T cells in bone marrow in tumor-bearing patients: an important role of bone marrow as a secondary lymphoid organ.

In tumor-bearing patients, tumor-associated antigen (TAA)-specific CTLs are spontaneously induced as a result of immune response to TAAs and play an important role in anti-tumor immunity. Wilms' tumor gene 1 (WT1) is overexpressed in various types of tumor and WT1 protein is a promising pan-TAA because of its high immunogenicity. In this study, to clarify the immune response to the WT1 antigen, WT1-specific CD8(+) T cells that were spontaneously induced in patients with solid tumor were comparatively analyzed in both bone marrow (BM) and peripheral blood (PB). WT1-specific CD8(+) T cells more frequently existed in BM than in PB, whereas frequencies of naïve (CCR7(+) CD45RA(+)), central memory (CCR7(+) CD45RA-), effector-memory (CCR7- CD45RA(-)), and effector (CCR7- CD45RA(+)) subsets were not significantly different between BM and PB. However, analysis of these subsets for the expression of CD57 and CD28, which were associated with differentiation, revealed that effector-memory and effector subsets of the WT1-specific CD8(+) T cells in BM had less differentiated phenotypes and more proliferative potential than those in PB. Furthermore, CD107a/b functional assay for WT1 peptide-specific cytotoxic potential and carboxyfluorescein diacetate succinimidyl ester dilution assay for WT1 peptide-specific proliferation also showed that WT1-specific CD8(+) T cells in BM were less cytotoxic and more proliferative in response to WT1 peptide than those in PB. These results implied that BM played an important role as a secondary lymphoid organ in tumor-bearing patients. Preferential residence of WT1-specific CD8(+) T cells in BM could be, at least in part, explained by higher expression of chemokine receptor CCR5, whose ligand was expressed on BM fibroblasts on the WT1-specific CD8(+) T cells in BM, compared to those in PB. These results should provide us with an insight into WT1-specific immune response in tumor-bearing patients and give us an idea of enhancement of clinical response in WT1 protein-targeted immunotherapy.

Biased usage of BV gene families of T-cell receptors of WT1 (Wilms' tumor gene)-specific CD8+ T cells in patients with myeloid malignancies.

WT1 (Wilms' tumor gene 1) protein is a potent pan-tumor-associated antigen (TAA) and WT1-specific cytotoxic T lymphocytes (WT1 tetramer(+) CD8(+) T cells) are spontaneously induced in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). We conducted a single-cell level comparative analysis of T-cell receptor beta-chain variable region (TCR-BV) gene families of a total of 1242 spontaneously induced WT1 tetramer(+) CD8(+) T cells in HLA-A*2402(+) patients with AML or MDS and those in healthy donors (HDs). This is the first report of direct usage analysis of TCR-BV gene families of individual TAA-specific CD8(+) T cells at single-cell level. Usage analysis using single-cell RT-PCR of TCR-BV gene families of individual FACS-sorted WT1 tetramer(+) CD8(+) T cells showed for the first time (i) that BVs 5, 6, 20, and 27 were commonly biased in both HDs and patients; (ii) that BV4 was commonly biased in HDs and MDS patients; (iii) that BV19 was commonly biased in the patients; and (iv) that BVs 7 and 28, BVs 9 and 15, and BVs 12 and 29 were specifically biased in HDs, AML, and MDS patients, respectively. However, statistical analysis of similarity among HD, AML, and MDS of individual usage frequencies of 24 kinds of TCR-BV gene families indicated that the usage frequencies of TCR-BV gene families in AML and MDS patients reflect those in HDs. These findings represent a novel insight for a better understanding of WT1-specific immune response.

Identification of a WT1 protein-derived peptide, WT1, 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 WT1(187) 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, WT1(187) 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.

A WT1 protein-derived, naturally processed 16-mer peptide, WT1(332), 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, WT1(332) (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 WT1(332) can induce WT1(332)-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 WT1(332)-specific CD4(+) T cell lines produced IFN-gamma but neither IL-4 nor IL-10 with WT1(332) stimulation, thus showing a Th1-type cytokine profile. Furthermore, HLA-DRB1*1501 or -DRB1*1502-restricted WT1(332)-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 WT1(332) is a naturally processed peptide. These results indicate that the natural peptide, WT1(332), is a promiscuous WT1-specific helper epitope. WT1(332) 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.

Clinical and immunologic responses to very low-dose vaccination with WT1 peptide (5 microg/body) in a patient with chronic myelomonocytic leukemia.

The wild-type Wilms tumor gene, WT1, is overexpressed in myelodysplastic syndrome (MDS) as well as acute myeloid leukemia. In a phase I clinical trial of biweekly vaccination with HLA-A*2402-restricted WT1 peptide for these malignancies, 2 patients with MDS developed severe leukocytopenia in association with a reduction in leukemic blast cells and levels of WT1 messenger RNA (mRNA) after only a single vaccination with 0.3 mg of WT1 peptide. These results indicated that the WT1-specific cytotoxic T-lymphocytes (CTLs) elicited by WT1 vaccination eradicated the WT1-expressing transformed stem or progenitor cells and that MDS patients with little normal hematopoiesis required a new strategy of WT1 vaccination to avoid severe leukocytopenia. We describe the first trial for a 57-year-old male patient with chronic myelomonocytic leukemia who was vaccinated biweekly with a small quantity (5 microg/body) of WT1 peptide. After the start of vaccination, the leukocyte and monocyte counts (13,780/microL and 1930/microL, respectively) gradually decreased to within the normal range in association with a reduction in the WT1 mRNA level. Simultaneously, the percentage of WT1-specific CTLs as measured by the HLA-WT1 tetramer assay increased. This case demonstrates for the first time that vaccination with as little as 5 microg of WT1 peptide can induce WT1-specific immune responses and resultant clinical responses.

Identification and characterization of a WT1 (Wilms Tumor Gene) protein-derived HLA-DRB1*0405-restricted 16-mer helper peptide that promotes the induction and activation of WT1-specific cytotoxic T lymphocytes.

Effective tumor vaccine may be required to induce both cytotoxic T lymphocyte (CTL) and CD4+ helper T-cell responses against tumor-associated antigens. CD4+ helper T cells that recognize HLA class II-restricted epitopes play a central role in the initiation and maintenance of antitumor immune responses. The Wilms tumor gene WT1 is overexpressed in both leukemias and solid tumors, and the WT1 protein was demonstrated to be an attractive target antigen for cancer immunotherapy. In this study, we identified a WT1 protein-derived 16-mer peptide, WT1(332)(KRYFKLSHLQMHSRKH), which was restricted with HLA-DRB1*0405, one of the most common HLA class II types in Japanese, as a helper epitope that could elicit WT1-specific CD4+ T-cell responses. We established a WT1(332)-specific CD4+ helper T-cell clone (E04.1), which could respond to both HLA-DRB1*0405-positive, WT1-expressing transformed hematopoietic cells and autologous dendritic cells pulsed with apoptosis-induced WT1-expressing cells, indicating that the WT1(332) was a naturally processed helper epitope. Stimulation of peripheral blood mononuclear cells with both the CTL epitope (WT1(235)) and the helper epitope (WT1(332)) in the presence of WT1(332)-specific TH1-type CD4+ T cell clone strikingly enhanced the induction and the functional activity of WT1(235)-specific CTLs compared with that of peripheral blood mononuclear cells with the WT1(235) alone. These results indicated that a helper epitope, WT1(332) should be useful for improvement of the efficacy of CTL epitope-based cancer vaccine targeting WT1 in the clinical setting.

AML1-ETO rapidly induces acute myeloblastic leukemia in cooperation with the Wilms tumor gene, WT1.

AML1-ETO, a chimeric gene frequently detected in acute myelogenous leukemia (AML), inhibits the differentiation of myeloid progenitors by suppressing genes associated with myeloid differentiation and increases the replating ability of clonogenic myeloid progenitors. However, AML1-ETO alone cannot induce AML and thus additional genetic events are required for the onset of AML. The Wilms tumor gene (WT1), which has been identified as the gene responsible for Wilms tumor, is expressed at high levels in almost all human leukemias. In this study, we have generated transgenic mice (WT1-Tg) that overexpress WT1 in hematopoietic cells to investigate the effects of WT1 on AML1-ETO-associated leukemogenesis. AML1-ETO-transduced bone marrow (BM) cells from WT1-Tg mice exhibited inhibition of myeloid differentiation at more immature stages and higher in vitro colony-forming ability compared with AML1-ETO-transduced BM cells from wild-type mice. Most importantly, all of the mice that received a transplant of AML1-ETO-transduced BM cells from the WT1-Tg mice rapidly developed AML. These results demonstrate that AML1-ETO may exert its leukemogenic function in cooperation with the expression of WT1.

Expansion of alpha-galactosylceramide-stimulated Valpha24+ NKT cells cultured in the absence of animal materials.

Valpha24+ NKT is an innate lymphocyte with potential antitumor activity. Clinical applications of Valpha24+ natural killer (NK) T cells, which are innate lymphocytes with potential antitumor activity, require their in vitro expansion. To avoid the potential dangers posed to patients by fetal bovine serum (FBS), the authors evaluated non-FBS culture conditions for the selective and efficient expansion of human Valpha24+ NKT cells. Mononuclear cells (MNCs) and plasma from the peripheral blood of normal healthy donors were used before and after G-CSF mobilization. MNCs and plasma separated from apheresis products were also used. MNCs were cultured for 12 days in AIM-V medium containing alpha-galactosylceramide (alpha-GalCer) (100 ng/mL) and IL-2 (100 U/mL) supplemented with FBS, autologous plasma, or autologous serum. The cultured cells were collected and their surface markers, intracellular cytokines, and cytotoxicity were evaluated. The highest expansion ratio for Valpha24+ NKT cells was obtained from G-CSF-mobilized MNCs cultured in medium containing 5% autologous plasma. Cultures containing MNCs and autologous plasma obtained before and after G-CSF mobilization had approximately 350-fold and 2,000-fold expansion ratios, respectively. These results suggest that G-CSF mobilization conferred a proliferative advantage to Valpha24+ NKT cells by modifying the biology of cells and plasma factors. Expanded Valpha24+ NKT cells retained their surface antigen expression and production of IFN-gamma and exhibited CD1d-independent cytotoxicity against tumor cells. Valpha24+ NKT cells can be efficiently expanded from G-CSF-mobilized peripheral blood MNCs in non-FBS culture conditions with alpha-GalCer and IL-2.

Freeze-thawing procedures have no influence on the phenotypic and functional development of dendritic cells generated from peripheral blood CD14+ monocytes.

Little is known about the potential influence of cryopreservation on the biologic activities of dendritic cells (DCs). In this study, we examined the effects of freeze-thawing on the phenotypic and functional development of human DCs obtained from granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood CD14+ cells. CD14+ cells were cultured, immediately or after freeze-thawing, with granulocyte-macrophage CSF and interleukin-4 for 9 days, and then with added tumor necrosis factor-alpha for another 3 days. For both fresh and freeze-thawed monocytes, immature DCs harvested on day 6 and mature DCs harvested on day 9 of culture were examined under the same conditions. Cells were compared with regard to their 1) capacities for antigen endocytosis and chemotactic migration (immature DCs), and 2) allogeneic mixed lymphocyte reaction and antigen-specific cytotoxic T lymphocyte responses (mature DCs). Freeze-thawing did not affect the viability or subsequent maturation of DCs at any stage of development. Furthermore, essentially no difference was observed in phenotype or function between cells generated from fresh or cryopreserved/thawed cells. Although this study design was limited with the use of fetal bovine serum, the observation still suggests that freeze-thawing does not affect viability, phenotype, subsequent maturation, or functions of DCs at any stage of maturation.

Increased expansion of V alpha 24+ T cells derived from G-CSF-mobilized peripheral blood stem cells as compared to peripheral blood mononuclear cells following alpha-galactosylceramide stimulation.

In the present study, unpurified peripheral blood mononuclear cells (PBMCs) from various sources, including steady-state blood (normal donors) and granulocyte colony-stimulating factor (G-CSF)-mobilized blood (cancer patients and normal donors) (G-PBSC), were cultured in RPMI-1640 in the presence of IL-2 and alpha-galactosylceramide (alpha-GalCer) to expand V alpha 24(+) T cells, and their expansion kinetics were compared. G-CSF-mobilized cells showed markedly higher expansion potential (350-fold expansion of V alpha 24(+) T cells, regardless of whether the cells were from cancer patients or normal donors) than steady-state cells (15-fold expansion, compared to the initial inoculums) (n = 5, P < 0.01). We also confirmed that the CD14(-) fraction of G-PBSCs contained a large number of precursors of V alpha 24(+) T cells, compared to PBSCs, as well as a large number of CD14(+) cells, which assist V alpha 24(+) T cell proliferation. Our simple and practical procedure, which eliminates complicated cell manipulation (including cell purification), produces efficient expansion of V alpha 24(+) T cells when G-CSF-mobilized blood cells are cultured with alpha-GalCer.

The role of PGE(2) in the differentiation of dendritic cells: how do dendritic cells influence T-cell polarization and chemokine receptor expression?

The role of prostaglandin E(2) (PGE(2)) in the function of dendritic cells (DCs), T-cell polarization, and expression of chemokine receptors was evaluated in human cells. Immature DCs were generated from peripheral blood CD14(+) cells using a combination of GM-CSF and interleukin-4 (IL-4) with or without PGE(2). On day 6, maturation of DCs was induced by the addition of tumor necrosis factor alpha with or without PGE(2). DCs harvested on day 6 (immature DCs) or day 9 (mature DCs) were examined using functional assays. In the presence of PGE(2), immature and mature DCs showed, phenotypically, a lower expression of CD1a and, functionally, a higher allostimulatory capacity at a high DC/T-cell ratio than control cells cultured in the absence of PGE(2). DCs cultured in the presence of PGE(2) induced the differentiation of naïve T cells toward a helper T-cell type 1 (Th1) response, which was independent of IL-12 secretion in the basal state despite a slightly lower interferon gamma secretion compared with control cells. However, the function of cytotoxicity-stimulating autologous T cells was not augmented by the addition of PGE(2). Immature DCs expressed the inflammatory chemokine receptors, CCR1 and CXCR4, but not CCR6, regardless of the presence or absence of PGE(2). Mature DCs expressed CCR7 equally, measured using a migration test and the measurement of calcium flux with macrophage inflammatory protein-3beta and reverse transcription-polymerase chain reaction assay in all of the groups. All of these findings suggest that PGE(2) affects the DC-promoted differentiation of naïve T cells to a Th1 response in the basal state, without affecting chemokine receptor expression on DCs.