Establishment of a novel NFAT-GFP reporter platform useful for the functional avidity maturation of HLA class II-restricted TCRs.

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.

Identification of mouse helper epitopes for WT1-specific CD4+ T cells.

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.

Identification of two distinct populations of WT1-specific cytotoxic T lymphocytes in co-vaccination of WT1 killer and helper peptides.

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.

Accumulation of HLA-DR4 in Colonic Epithelial Cells Causes Severe Colitis in Homozygous HLA-DR4 Transgenic Mice.

BACKGROUND: Homozygous HLA-DR4/I-E transgenic mice (tgm) spontaneously developed colitis similar to human ulcerative colitis. We explored whether endoplasmic reticulum stress in colonic epithelial cells due to overexpression of HLA-DR4/I-E was involved in the pathogenesis of colitis. METHODS: Major histocompatibility complex class II transactivator-knockout (CIITAKO) background tgm were established to test the involvement of HLA-DR4/I-E expression in the pathogenesis of colitis. Histological and cellular analyses were performed and the effect of oral administration of the molecular chaperone tauroursodeoxycholic acid (TUDCA) and antibiotics were investigated. IgA content of feces and serum and presence of IgA-coated fecal bacteria were also investigated. RESULTS: Aberrantly accumulated HLA-DR4/I-E molecules in colonic epithelial cells were observed only in the colitic homozygous tgm, which was accompanied by upregulation of the endoplasmic reticulum stress marker Binding immunoglobulin protein (BiP) and reduced mucus. Homozygous tgm with CIITAKO, and thus absent of HLA-DR4/I-E expression, did not develop colitis. Oral administration of TUDCA to homozygotes reduced HLA-DR4/I-E and BiP expression in colonic epithelial cells and restored the barrier function of the intestinal tract. The IgA content of feces and serum, and numbers of IgA-coated fecal bacteria were higher in the colitic tgm, and antibiotic administration suppressed the expression of HLA-DR4/I-E and colitis. CONCLUSIONS: The pathogenesis of the colitis observed in the homozygous tgm was likely due to endoplasmic reticulum stress, resulting in goblet cell damage and compromised mucus production in the colonic epithelial cells in which HLA-DR4/I-E molecules were heavily accumulated. Commensal bacteria seemed to be involved in the accumulation of HLA-DR4/I-E, leading to development of the colitis.

A new peptide vaccine OCV-501: in vitro pharmacology and phase 1 study in patients with acute myeloid leukemia.

Wilms' tumor 1 (WT1) is a promising target of new immunotherapies for acute myeloid leukemia (AML) as well as for other cancers. OCV-501 is a helper peptide derived from the WT1 protein. OCV-501 induced OCV-501-specific Type 1 T-helper (Th1) responses dose-dependently and stimulated helper activity of the specific Th1 cells in peripheral blood mononuclear cells from healthy donors. OCV-501 also enhanced the increase in WT1-killer peptide-specific cytotoxic T lymphocytes. OCV-501 stimulated the OCV-501-specific Th1 clones in an HLA class-II restricted manner and formed a complex with HLA class-II protein. OCV-501-specific Th1 clones demonstrated significant OCV-501-specific cytolytic activity against OCV-501-pulsed B-lymphoblastoid cell line cells. Based on the pre-clinical results, phase 1 clinical trial was conducted. The result of this trial suggested that the subcutaneous administration of OCV-501 once weekly for 4 weeks at doses of 0.3, 1, and 3 mg in older patients with AML during complete remission was safe and well tolerated. The maximum tolerated dose was considered to be ≥3 mg. Of the nine subjects enrolled, neither relapse nor blast cells were observed during the study. Immunological responses were observed in OCV-501-specific delayed-type hypersensitivity test. This trial was registered at http://www.clinicaltrials.gov as NCT 01440920.

Transduction of a novel HLA-DRB1*04:05-restricted, WT1-specific TCR gene into human CD4+ T cells confers killing activity against human leukemia cells.

BACKGROUND/AIM: Wilms' tumor gene 1 (WT1) product is a pan-tumor-associated antigen. We previously identified WT1 protein-derived promiscuous helper peptide, WT1332. Therefore, isolation and characterization of the WT1332-specific T-cell receptors (TCRs) are useful to develop broadly applicable TCR gene-based adoptive immunotherapy. MATERIALS AND METHODS: A novel HLA-DRB1*04:05-restricted WT1332-specific TCR gene was cloned and transduced into human CD4+ T-cells by using a lentiviral vector. RESULTS: The WT1332-specific TCR-transduced CD4+ T-cells showed strong proliferation and Th1-cytokine production in an HLA-DRB1*04:05-restricted, WT1332-specific manner. Furthermore, the WT1332-specific TCR-transduced CD4+ T-cells could lyse HLA-DRB1*04:05-positive, WT1-expressing leukemia cells in vitro. CONCLUSION: The novel TCR gene cloned here should be a promising tool to develop adoptive immunotherapy of WT1332-specific TCR-transduced CD4+ T-cells for the treatment of WT1-expressing cancer, such as leukemia.

Establishment of HLA-DR4 transgenic mice for the identification of CD4+ T cell epitopes of tumor-associated antigens.

Reports have shown that activation of tumor-specific CD4(+) helper T (Th) cells is crucial for effective anti-tumor immunity and identification of Th-cell epitopes is critical for peptide vaccine-based cancer immunotherapy. Although computer algorithms are available to predict peptides with high binding affinity to a specific HLA class II molecule, the ability of those peptides to induce Th-cell responses must be evaluated. We have established HLA-DR4 (HLA-DRA*01:01/HLA-DRB1*04:05) transgenic mice (Tgm), since this HLA-DR allele is most frequent (13.6%) in Japanese population, to evaluate HLA-DR4-restricted Th-cell responses to tumor-associated antigen (TAA)-derived peptides predicted to bind to HLA-DR4. To avoid weak binding between mouse CD4 and HLA-DR4, Tgm were designed to express chimeric HLA-DR4/I-E(d), where I-E(d) α1 and ß1 domains were replaced with those from HLA-DR4. Th cells isolated from Tgm immunized with adjuvant and HLA-DR4-binding cytomegalovirus-derived peptide proliferated when stimulated with peptide-pulsed HLA-DR4-transduced mouse L cells, indicating chimeric HLA-DR4/I-E(d) has equivalent antigen presenting capacity to HLA-DR4. Immunization with CDCA155-78 peptide, a computer algorithm-predicted HLA-DR4-binding peptide derived from TAA CDCA1, successfully induced Th-cell responses in Tgm, while immunization of HLA-DR4-binding Wilms' tumor 1 antigen-derived peptide with identical amino acid sequence to mouse ortholog failed. This was overcome by using peptide-pulsed syngeneic bone marrow-derived dendritic cells (BM-DC) followed by immunization with peptide/CFA booster. BM-DC-based immunization of KIF20A494-517 peptide from another TAA KIF20A, with an almost identical HLA-binding core amino acid sequence to mouse ortholog, successfully induced Th-cell responses in Tgm. Notably, both CDCA155-78 and KIF20A494-517 peptides induced human Th-cell responses in PBMCs from HLA-DR4-positive donors. Finally, an HLA-DR4 binding DEPDC1191-213 peptide from a new TAA DEPDC1 overexpressed in bladder cancer induced strong Th-cell responses both in Tgm and in PBMCs from an HLA-DR4-positive donor. Thus, the HLA-DR4 Tgm combined with computer algorithm was useful for preliminary screening of candidate peptides for vaccination.

Gene expression analysis during platelet-like particle production in phorbol myristate acetate-treated MEG-01 cells.

A comprehensive gene-expression analysis during platelet (PLT) production from megakaryocytes may give important information on genes involved in the PLT production process. However, the low abundance of primary megakaryocytes makes the gene expression analysis difficult. Therefore, we employed MEG-01 cells, a human megakaryocytic cell line, and confirmed that the cell line produces PLT-like particles by treatment with phorbol myristate acetate (PMA). After treatment of MEG-01 cells with PMA for 8 or 24 h, comprehensive gene expression analysis was carried out using a microarray and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). From the microarray analysis, 141 genes were up-regulated (>2-fold) and 164 genes were down-regulated (<1/2-fold). However, known PLT-related genes were not included in the up- or down-regulated genes. On the other hand, RT-PCR analysis detected increased expression of beta1-tubulin, CD62P, gpIbalpha and gpIII, which are related to PLT function and megakaryocyte differentiation, following PMA treatment for 24 h. These results indicate that the MEG-01 cell may be an alternative model system to study the process of human PLT production from megakaryocytes. The gene-expression analysis might be a powerful tool for identifying genes related to PLT production, if the experimental conditions are optimized.

Lactoferrin inhibits platelet production from human megakaryocytes in vitro.

The mechanism of megakaryopoiesis, proplatelet formation (PPF) and platelet (PLT) production is not fully elucidated. Lactoferrin (LF) has been reported to have many biological functions including cell proliferation and differentiation, and the LF receptor is present on megakaryocytic cells. In the present study, we examined the effect of human LF (hLF) on PLT production from primary megakaryocytes (MKs). At first, we developed a PLT production system derived from human CD34+ cells by thrombopoietin (TPO) stimulation. Because the number of proplatelets, PLTs and CD41+ MKs was remarkably increased after day 5, we employed the TPO-induced CD34+ cells on day 5. Then, the effect of hLF on PLT production from human primary MKs was examined. In the range of 3-30 micrg/ml, hLF significantly inhibited PLT production up to about 60%. However, it did not significantly change the intensity of CD41 expression in MKs and the ploidy of MKs. In addition, it did not inhibit MK progenitors. These results suggest that LF directly inhibits PLT production from matured MKs, but does not inhibit megakaryopoiesis, including proliferation/maturation processes.

CD41+/CD45+ cells without acetylcholinesterase activity are immature and a major megakaryocytic population in murine bone marrow.

Murine megakaryocytes (MKs) are defined by CD41/CD61 expression and acetylcholinesterase (AChE) activity; however, their stages of differentiation in bone marrow (BM) have not been fully elucidated. In murine lineage-negative (Lin(-))/CD45(+) BM cells, we found CD41(+) MKs without AChE activity (AChE(-)) except for CD41(++) MKs with AChE activity (AChE(+)), in which CD61 expression was similar to their CD41 level. Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs could differentiate into AChE(+), with an accompanying increase in CD41/CD61 during in vitro culture. Both proplatelet formation (PPF) and platelet (PLT) production for Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs were observed later than for Lin(-)/CD41(++)/CD45(+)/AChE(+) MKs, whereas MK progenitors were scarcely detected in both subpopulations. GeneChip and semiquantitative polymerase chain reaction analyses revealed that the Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs are assigned at the stage between the progenitor and PPF preparation phases in respect to the many MK/PLT-specific gene expressions, including beta1-tubulin. In normal mice, the number of Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs was 100 times higher than that of AChE(+) MKs in BM. When MK destruction and consequent thrombocytopenia were caused by an antitumor agent, mitomycin-C, Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs led to an increase in AChE(+) MKs and subsequent PLT recovery with interleukin-11 administration. It was concluded that MKs in murine BM at least in part consist of immature Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs and more differentiated Lin(-)/CD41(++)/CD45(+)/AChE(+) MKs. Immature Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs are a major MK population compared with AChE(+) MKs in BM and play an important role in rapid PLT recovery in vivo.

Efficient gene expression in megakaryocytic cell line using nucleofection.

To clarify the mechanism of platelet production from megakaryocytes, expression of target proteins by gene transfection was examined using various gene delivery techniques. Transfection into hematopoietic cells, including megakaryocytes, by conventional gene delivery techniques such as electroporation and lipofection are known to be difficult. In this study, in addition to electroporation and lipofection, we tested other gene-transfer methods (nucleofection, transfection using inactivated virus envelope, and transferrin-linked cationic polymer) with the green fluorescent protein (GFP) gene into the human megakaryocytic cell line MEG-01. We found that nucleofection, which uses a combination of special electrical parameters and specific solutions, was the best, judging from the expression ratio of GFP-positive cells (approximately 70% of cells) and low toxicity. The efficiency of GFP expression was not related to the amount of pDNA delivered into the MEG-01 cells. To verify the utility of nucleofection, the thrombopoietin (TPO) receptor c-mpl was transfected into MEG-01 cells. Transfected cells showed a higher responsiveness to TPO than mock-transfected MEG-01 cells. We propose that nucleofection is a useful method for transfecting target genes to megakaryocytic cells when addressing the mechanism of platelet production.