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CD4+ T cells that recognize antigenic peptides presented on HLA class II are essential for inducing an optimal anti-tumor immune response, and adoptive transfer of tumor antigen-specific TCR-transduced CD4+ T cells with high responsiveness against tumor is a promising strategy for cancer treatment. Whereas a precise evaluation method of functional avidity, an indicator of T cell responsiveness against tumors, has been established for HLA class I-restricted TCRs, it remains unestablished for HLA class II-restricted TCRs. In this study, we generated a novel platform cell line, CD4-2D3, in which GFP reporter was expressed by NFAT activation via TCR signaling, for correctly evaluating functional avidity of HLA class II-restricted TCRs. Furthermore, using this platform cell line, we succeeded in maturating functional avidity of an HLA class II-restricted TCR specific for a WT1-derived helper peptide by substituting amino acids in complementarity determining region 3 (CDR3) of the TCR. Importantly, we demonstrated that transduction of an avidity-maturated TCR conferred strong cytotoxicity against WT1-expressing leukemia cells on CD4+ T cells, compared to that of its original TCR. Thus, CD4-2D3 cell line should be useful not only to evaluate TCR functional avidity in HLA class II-restricted TCRs but also to screen appropriate TCRs for clinical applications such as cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Simple Summary: Therapeutic options for rare cancers are frequently limited and less effective than for common cancers. Therefore, novel therapeutic strategies to treat rare cancers are urgently required. Our clinical study on rare cancers showed that the biweekly WT1 Trio peptide vaccine comprising two WT1-cytotoxic T lymphocyte (CTL)-peptides and one WT1-helper T lymphocyte-peptide induced more robust immune responses targeting WT1 than the weekly WT1-CTL peptide (WT1-235) vaccine. In addition, the safety of WT1 Trio was confirmed without severe treatment-related adverse events, except grade 3 myasthenia gravis-like symptoms observed in a patient with thymic cancer (TC). Fifteen (33.3%) of the 45 patients with recurrent or advanced rare cancers, including malignant glioma, soft-tissue sarcoma, TC, and malignant pleural mesothelioma, achieved stable disease after 3 months of protocol treatment. Therefore, since WT1 is widely overexpressed in rare cancers, WT1-targeted immunotherapy may be a therapeutic strategy for rare cancers. No standard treatment has been established for most rare cancers. Here, we report a clinical trial of a biweekly WT1 tri-peptide-based vaccine for recurrent or advanced rare cancers. Due to the insufficient number of patients available for a traditional clinical trial, the trial was designed for rare cancers expressing shared target molecule WT1. The recruitment criteria included WT1-expressing tumors as well as HLA-A*24:02 or 02:01. The primary endpoints were immunoglobulin G (IgG) antibody (Ab) production against the WT1-235 cytotoxic T lymphocyte (CTL) epitope and delayed-type hypersensitivity (DTH) skin reactions to targeted WT1 CTL epitopes. The secondary endpoints were safety and clinical efficacy. Forty-five patients received WT1 Trio, and 25 (55.6%) completed the 3-month protocol treatment. WT1-235 IgG Ab was positive in 88.0% of patients treated with WT1 Trio at 3 months, significantly higher than 62.5% of the weekly WT1-235 CTL peptide vaccine. The DTH positivity rate in WT1 Trio was 62.9%, which was not significantly different from 60.7% in the WT1-235 CTL peptide vaccine. The WT1 Trio safety was confirmed without severe treatment-related adverse events, except grade 3 myasthenia gravis-like symptoms observed in a patient with thymic cancer. Fifteen (33.3%) patients achieved stable disease after 3 months of treatment. In conclusion, the biweekly WT1 Trio vaccine containing the WT1-332 helper T lymphocyte peptide induced more robust immune responses targeting WT1 than the weekly WT1-235 CTL peptide vaccine. Therefore, WT1-targeted immunotherapy may be a potential therapeutic strategy for rare cancers. [ABSTRACT FROM AUTHOR]

Wilms' tumor 1 (WT1) is a promising tumor-associated antigen for cancer immunotherapy. We developed an oral protein vaccine platform composed of WT1-anchored, genetically engineered Bifidobacterium longum (B. longum) and conducted an in vivo study in mice to examine its anticancer activity. Mice were orally treated with phosphate-buffered saline, wild-type B. longum105-A, B. longum 2012 displaying only galacto-N-biose/lacto-N-biose I-binding protein (GLBP), and WT1 protein- and GLBP-expressing B. longum 420. Tumor size reduced significantly in the B. longum 420 group than in the B. longum 105-A and 2012 groups (P < 0.00 l each), indicating B. longum 420's antitumor activity via WT1-specific immune responses. CD8+ T cells played a major role in the antitumor activity of B. longum 420. The proportion of CD103+CD11b+CD11c+ dendritic cells (DCs) increased in the Peyer's patches (PPs) from mice in the B. longum 420 group, indicating the definite activation of DCs. In the PPs, the number and proportion of CD8+ T cells capable of producing interferon-gamma were significantly greater in the B. longum 420 group than in the B. longum 2012 group (P < 0.05 or < 0.01). The production of WT1-specific IgG antibody was significantly higher in the B. longum 420 group than in the 2012 group (P < 0.05). The B. longum 420 group showed the most intense intratumoral infiltration of CD4+ and CD8+ T cells primed by activated DCs in the PPs of mice in the B. longum 420 group. Our findings provide insights into a novel, intestinal bacterium-based, cancer immunotherapy through intestinal immunity. [ABSTRACT FROM AUTHOR]

Helper T lymphocytes (HTLs) play a central role in cancer immunity because they can not only help the induction and proliferation of cytotoxic T lymphocytes (CTLs) but also their differentiation into cytotoxic CD4+ T cells and directly kill the target cells.This study describes the identification of three novel mouse Th epitope peptides, WT135-52, WT186-102 and WT1294-312, derived from WT1 protein, which is the most potent tumor-associated antigen. Compared to immunization with WT1 CTL peptide alone, immunization with the addition of these WT1-specific Th peptides strongly induced WT1-specific CTLs, continued to maintain them, and efficiently rejected the challenge of WT1-expressing tumor cells. Importantly, the majority of WT1-specific CTLs induced by the co-immunization with WT1 CTL and the WT1-specific Th peptides were CD44+CD62L− effector memory CD8+ T cells, which played a central role in tumor rejection. Establishment of mouse models suitable for the analysis of the detailed mechanism of these functions of HTLs is very important. These results clearly showed that WT1-specific HTLs perform an essential function in WT1-specific tumor immunity. Therefore, the WT1-specific Th peptides identified here should make a major contribution to elucidation of the mutual roles of WT1-specific CTLs and HTLs in cancer immunity in in vivo mouse models. [ABSTRACT FROM AUTHOR]

Simultaneous induction of tumor antigen-specific cytotoxic T lymphocytes (CTLs) and helper T lymphocytes (HTLs) is required for an optimal anti-tumor immune response. WT1332, a 16-mer WT1-derived helper peptide, induce HTLs in an HLA class II-restricted manner and enhance the induction of WT1-specific CTLs in vitro. However, in vivo immune reaction to WT1332 vaccination in tumor-bearing patients remained unclear. Here, a striking difference in WT1-specific T cell responses was shown between WT1 CTL + WT1 helper peptide and WT1 CTL peptide vaccines in patients with recurrent glioma. WT1-specific CTLs were more strongly induced in the patients who were immunized with WT1 CTL + WT1 helper peptide vaccine, compared to those who were immunized with WT1 CTL vaccine alone. Importantly, a clear correlation was demonstrated between WT1-specific CTL and WT1332-specific HTL responses. Interestingly, two novel distinct populations of WT1-tetramerlow WT1-TCRlow CD5low and WT1-tetramerhigh WT1-TCRhigh CD5high CTLs were dominantly detected in WT1 CTL + WT1 helper peptide vaccine. Although natural WT1 peptide-reactive CTLs in the latter population were evidently less than those in the former population, the latter population showed natural WT1 peptide-specific proliferation capacity comparable to the former population, suggesting that the latter population highly expressing CD5, a marker of resistance to activation-induced cell death, should strongly expand and persist for a long time in patients. These results demonstrated the advantage of WT1 helper peptide vaccine for the enhancement of WT1-specific CTL induction by WT1 CTL peptide vaccine. [ABSTRACT FROM AUTHOR]

To improve cancer immunotherapy, it is important to understand how tumor cells counteract immune-surveillance. In this study, we sought to identify cell-surface molecules associated with resistance of leukemia cells to cytotoxic T cell (CTL)-mediated cytolysis. To this end, we first established thousands of monoclonal antibodies (mAbs) that react with MLL/AF9 mouse leukemia cells. Only two of these mAbs, designated R54 and B2, bound preferentially to leukemia cells resistant to cytolysis by a tumor cell antigen–specific CTLs. The antigens recognized by these mAbs were identified by expression cloning as the same protein, CD43, although their binding patterns to subsets of hematopoietic cells differed significantly from each other and from a pre-existing pan-CD43 mAb, S11. The epitopes of R54 and B2, but not S11, were sialidase-sensitive and expressed at various levels on leukemia cells, suggesting that binding of R54 or B2 is associated with the glycosylation status of CD43. R54high leukemia cells, which are likely to express sialic acid-rich CD43, were highly resistant to CTL-mediated cytolysis. In addition, loss of CD43 in leukemia cells or neuraminidase treatment of leukemia cells sensitized leukemia cells to CTL-mediated cell lysis. These results suggest that sialic acid-rich CD43, which harbors multiple sialic acid residues that impart a net negative surface charge, protects leukemia cells from CTL-mediated cell lysis. Furthermore, R54high or B2high leukemia cells preferentially survived in vivo in the presence of adaptive immunity. Taken together, these results suggest that the glycosylation status of CD43 on leukemia is associated with sensitivity to CTL-mediated cytolysis in vitro and in vivo. Thus, regulation of CD43 glycosylation is a potential strategy for enhancing CTL-mediated immunotherapy. [ABSTRACT FROM AUTHOR]

Th17 plays important roles in the pathogenesis of various inflammatory and autoimmune diseases. Although the importance of Th17 in tumor immunity has also been suggested, precise roles of tumor-associated antigen-specific Th17 still remain poorly understood, especially in humans. We previously identified WT1, a 16-mer helper epitope derived from tumor-associated antigen Wilms' tumor gene 1 (WT1) product, and WT1-specific Th1 clones were established. In the present study, WT1-specific Th17 clones were established by the stimulation of peripheral blood mononuclear cells with the WT1 helper peptide under human Th17-polarizing conditions. The WT1-specific Th17 clone exhibited the helper function for proliferation of conventional CD4 T cells in the antigenic stimulation-specific manner. This is the first report of establishment of functional Th17 clones with both antigen (WT1) specificity and antigen-specific helper activity. Th17 clones established here and the method to establish antigen-specific Th17 clones should be a useful tool to further analyze the roles of human Th17 in tumor immunity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Wilms’ tumor gene WT1 is expressed in various kinds of cancers. Human WT1-specific cytotoxic T lymphocytes (CTLs) were generated, and mice immunized with WT1 peptide rejected challenges by WT1-expressing cancer cells without auto-aggression to normal organs. Furthermore, WT1 antibodies and WT1-specific CTLs were detected in cancer patients, indicating that WT1 protein was immunogenic. These findings provided us with the rationale for cancer immunotherapy targeting WT1. Clinical trials of WT1 peptide vaccination for cancer patients were started, and WT1 vaccination-related immunological responses and clinical responses, including reduction of leukemic cells, reduction of M-protein amount in myeloma, and shrinkage of solid cancer, were observed. Valuable information about immune responses against tumor antigens can be obtained by the analysis of samples from the vaccinated patients, which should lead to further improvement of cancer vaccine. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: To induce and activate tumor-associated antigen-specific cytotoxic T lymphocytes (CTLs) for cancer immunity, it is important not only to select potent CTL epitopes but also to combine them with appropriate immunopotentiating agents. Here we investigated whether tumor immunity induced by WT1 peptide vaccination could be enhanced by IFN-β. For the experimental group, C57BL/6 mice were twice pre-treated with WT1 peptide vaccine, implanted with WT1-expressing C1498 cells, and treated four times with WT1 peptide vaccine at one-week intervals. During the vaccination period, IFN-β was injected three times a week. Mice in control groups were treated with WT1 peptide alone, IFN-β alone, or PBS alone. The mice in the experimental group rejected tumor cells and survived significantly longer than mice in the control groups. The overall survival on day 75 was 40% for the mice treated with WT1 peptide+IFN-β, while it was 7, 7, and 0% for those treated with WT1 peptide alone, IFN-β alone or PBS alone, respectively. Induction of WT1-specific CTLs and enhancement of NK activity were detected in splenocytes from mice in the experimental group. Furthermore, administration of IFN-β enhanced expression of MHC class I molecules on the implanted tumor cells. In conclusion, our results showed that co-administration of WT1 peptide+IFN-β enhanced tumor immunity mainly through the induction of WT1-specific CTLs, enhancement of NK activity, and promotion of MHC class I expression on the tumor cells. WT1 peptide vaccination combined with IFN-β administration can thus be expected to enhance the clinical efficacy of WT1 immunotherapy. [Copyright &y& Elsevier]

Activation-induced cell death (AICD) of lymphocytes is an apoptotic pathway involved in the control of T-cell homeostasis. The magnitude of graft-vs.-host disease (GVHD) following allogeneic hematopoietic cell transplantation may be attenuated by the enhancement of AICD. The aim of the present study was to clarify the effect of T cell dose upon the fate (proliferation or apoptosis) of individual activated T cells in a murine GVHD model. To this end, we investigated the kinetics of the proliferation and apoptosis of donor T cells in recipient spleens in the early stage of a fully major histocompatibility complex (MHC)mismatched murine transplantation model from C57BL/6 (H-2[sup b]) to lethally-irradiated (8.5 Gy) BALB/c (H-2[sup d]) mice. To track the behavior of alloreactive lymphocytes in vivo, we used the fluorescent cytoplasmic dye carboxyfluorescein diacetate succinimidyl ester in combination with flow cytometry. Engraftment of donor T cells to recipient spleens was almost completed within 24 h after transfer. After that, at higher doses of transferred cells, the donor T cells actively divided for up to 72 h resulting in a 30fold increase in cell number at the maximum cell dose (2.0 × 10[sup 7]). As the transferred cell dose decreased, the proliferation of T cells tended to be suppressed. At cell doses of 0.5 × 10[sup 7] or less, the proliferation of T cells was profoundly suppressed, ultimately resulting in little proliferation of donor T cells observed from 24 to 72 h at the minimum cell dose (0.1 × 10[sup 7]). The frequency of Annexin-V-positive cells was found to increase gradually as the transferred cell dose decreased. Thus, an increase in apoptotic events appeared to play an important role in the suppression of the proliferation of T cells at lower splenocyte doses. Further analyses revealed that Fas ligand (FasL)-positive T cells were observed exclusively among T cells that divided at least 5 times, and that all of them were... [ABSTRACT FROM AUTHOR]