ALBERT

Description: The protective immunity afforded by CD8+ T cells against blood-stage malaria remains controversial because no MHC class I molecules are displayed on parasite-infected human erythrocytes. We recently reported that rodent malaria parasites infect erythroblasts that express major histocompatibility complex (MHC) class I antigens, which are recognized by CD8+ T cells. In this study, we demonstrate that the cytotoxic activity of CD8+ T cells contributes to the protection of mice against blood-stage malaria in a Fas ligand (FasL)-dependent manner. Erythroblasts infected with malarial parasites express the death receptor Fas. CD8+ T cells induce the externalization of phosphatidylserine (PS) on the infected erythroblasts in a cell-to-cell contact-dependent manner. PS enhances the engulfment of the infected erythroid cells by phagocytes. As a PS receptor, T-cell immunoglobulin-domain and mucin-domain-containing molecule 4 (Tim-4) contributes to the phagocytosis of malaria-parasite-infected cells. Our findings provide insight into the molecular mechanisms underlying the protective immunity exerted by CD8+ T cells in collaboration with phagocytes.

Description: Abstract 3763 Introduction: tRNase ZL is one of the enzymes for tRNA maturation and removes a 3′ trailer of pre-tRNA by recognizing four leaves-clover-like structure of pre-tRNA. In addition, we found that tRNase ZL can recognize 12-bases hairpin structures and cleaves 3′ portion of them. Furthermore, we have demonstrated that tRNase ZL could cleave 3′ portion of target mRNA by recognizing a complex consisting of both the target mRNA and artificially designed small guide RNA (sgRNA) which possesses the complementary sequences with target RNA and form a four leaves-clover-like structure or 12-bases hairpin structures with target mRNA. Currently, sgRNA is divided into five categories; 3′-truncated tRNA (form a four leaves-clover-like structure with target mRNA), 5′-half tRNA (form a half of four leaves-clover-like structure with target mRNA), 14-nucleotides linear type sgRNA (form a 14-bases stem with target mRNA), hook type sgRNA (containing a single loop structure and form 12-bases hairpin structure with target mRNA) and heptamer sgRNA (7-bases RNA structure complementary to target mRNA and form a 12-bases hairpin structure with target mRNA). The use of sgRNA, especially heptamer sgRNA, to knockdown specific mRNAs has several advantages over long form RNAs (antisense, shRNA or siRNA); 1) heptamer sgRNA can be introduced into cells without any transfection operation, 2) less off-target effect, 3 less interferon responses, 4) the synthesis of heptamer sgRNA is inexpensive than that of long form RNAs. Thus, gene silencing methods using tRNase ZL and sgRNA are presumed to induce effective and specific target RNA inhibition. In the present study, in order to explore the application of sgRNA (especially heptamer sgRNA) as novel nucleic acid medicine, we investigated the effects of heptamer sgRNA targeting WT1 gene (WT1sgRNA) and PRMAE gene (PRAMEsgRNA) on apoptosis, proliferation and mRNA suppression in leukemic cell lines. Materials and Methods: The 5′-phosphorylated 2′-O-methyl heptamer sgRNAs, which were designed to form 12-bases hairpin structure with target mRNA of leukemia associated antigens (LAA) were synthesized and purified by high-performance liquid chromatography. The leukemic cell lines (HL60, C2F8 and U937) were cultured with heptamer sgRNA at the concentration of 1 mM. Introduction of heptamer sgRNA into cells was investigated by using FITC-conjugated heptamer sgRNA and evaluated by flow cytometry and fluorescence microscopy. Quantification of target mRNA was determined by real-time quantitative PCR. Effects of heptamer sgRNA on apoptosis of leukemic cells were evaluated by flow cytometry. Inhibition of cell proliferation was evaluated by TetraColor ONE reagent. Results: Heptamer sgRNA could be introduced into cells by just adding them to culture medium by both flow cytometry and fluorescent microscopy. Expression level of WT1 and PRAME mRNA in HL60 cells was reduced by culturing HL60 cells with WT1sgRNA and PRAMEsgRNA (up to 75% and 30% reduction, respectively) compared to control EGFPsgRNA (estimated as 0% reduction). Likewise, WT1sgRNA suppressed WT1 mRNA expression in C2F8 cells by just adding WT1sgRNA to culture medium (30% reduction). In addition, WT1sgRNA and PRAMEsgRNA could induce apoptosis in HL60 cells but not in WT1(–)PRAME(–) U937 cells. Control EGFPsgRNA did not induce apoptosis. WT1sgRNA and PRAMEsgRNA could inhibit cell proliferation in HL60 cells (70% and 40% inhibition) respectively, compared to control EGFPsgRNA (estimated as 0% inhibition). WT1sgRNA could also inhibit cell proliferation in C2F8 cells (70% inhibition). Conclusion: These data indicate that WT1sgRNA and PRAMEsgRNA specifically bind to target mRNA and forms 12-bases hairpin structure with target mRNA, which complex is recognized by tRNase ZL. Then 3′ portion of target mRNA is cleaved by tRNase ZL by target-specific manner. Taken together, these findings suggested a possibility that oncogene-targeting sgRNA (especially heptamer sgRNA) could be applied as a novel nucleic acid medicine against leukemia and other tumors. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4877 It is important to understand the mechanisms of naturally acquired immunity to malaria for the development of effective malaria vaccines. We have demonstrated that γδT cells expanded in the peripheral blood of the falciparum malaria patients in Thailand but did not expand in patients living in malaria endemic areas of Laos. However, the percentage of Vγ9-T cells, a γδT cell subset, increased in the Laos patients. The levels of naturally acquired antibodies to crude P. falciparum (Pf) antigens also increased with an age dependent manner in individuals living in endemic areas of Laos. In this study, we further investigated the role of Vγ9-T cells in naturally acquired immunity to the falciparum malaria. The peripheral blood lymphocytes (PBLs) and plasma obtained from hospitalized uncomplicated falciparum malaria patients (UMPs) and severe falciparum malaria patients (SMPs) in Thailand and from non-hospitalized UMPs living in endemic areas of Laos were analyzed. The plasma levels of IL-10, which is anti-inflammatory cytokine and associated with antibody production from B cells, were elevated in both hospitalized and non-hospitalized falciparum malaria patients. There was a correlation between the levels of IL-10 and the percentage of Vγ9-T cells in γδT cells. IL-10 and Pf specific antibodies were detected only in culture supernatant of PBLs from non-hospitalized UMPs in the presence of IL-2 for 2 wks. These results indicate that Vγ9-T cells may contribute to acquiring natural immunity to malaria. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4253 Cytotoxic T lymphocytes (CTLs) are presumed to kill the relevant antigen-expressing leukemia cells including leukemic stem cells, which display intrinsic resistance against tyrosine kinase inhibitors such as imatinib in CML patients. In order to clarify the safety and effectiveness of WT1 peptide vaccination for the patients with CML, we started WT1 peptide vaccination in combination with imatinib therapy for a CML patient who could not acquire a major molecular response by the administration of imatinib. In addition, we tried to evaluate the kinetics of WT1-specific CTLs in peripheral blood (PB) during WT1 peptide vaccination. A 51 years-old male with CML in chronic phase had been treated with 400 mg imatinib for 4 years. BCR-ABL transcripts decreased transiently to less than 700 copies in 1 μg RNA (3-log reduction: 300 copies in 1 μg RNA extracted from PB cells; median in our laboratory) but gradually increased to more than 1,000 copies thereafter. Since the patient was HLA-A*2402+ and an informed consent was obtained, HLA-A*2402-restricted 9mer WT1 peptides (CYTWNQMNL; a.a. 235-243), which had been identified to possess an anti-tumor immunogenicity, were administered subcutaneously at the dose of 1 mg/day every 2 weeks in combination with 400 mg imatinib for first 5 months and thereafter every 4 weeks for 12 months. No adverse effects due to WT1 peptide vaccination was observed except for skin induration and redness at the sites of WT1 peptide injection. Although BCR-ABL transcripts increased up to more than 2,000 copies after transient decrease to less than 1,000 copies by the administration of WT1 peptides every 2 weeks, the transcripts have decreased to less than 500 copies by the administration of WT1 peptides every 4 weeks. The appearance of WT1-specific CTLs in PB was confirmed by evaluating the frequency of MHC/WT1 tetramer+CD8+ T cells by using MLPC (mixed lymphocyte peptide culture). MLPC was performed by culturing 1-3 ×105 PB mononuclear cells (MNCs) in 200 l of 5% autologous serum-containing medium with 10 μg WT1 peptides in 96 well plates. Frequency of MHC/WT1 tetramer+ cells in PB-CD8+ cells was calculated by the following formula. Number of wells containing a lump of tetramer+CD8+ cells / [(Number of PB-MNCs seeded in a well of MLPC) x (total number of wells for MLPC) x (ratio of PB-CD8+ cells in PB-MNCs)]. Although WT1-specific CTLs were not detected in PB before WT1 peptide vaccination, the CTLs appeared after the second vaccination and maintained at the level of nearly 15/106 CD8+ cells thereafter. The MHC/WT1 tetramer+ cells showed cytotoxicity against only leukemia cells expressing WT1 and HLA-A*2402. The cytotoxicity was blocked clearly by adding unlabeled cold target cells in cold inhibition test and blocked also by antibodies against MHC class I but not by antibodies against MHC class II. In vitro study demonstrated that the stimulation with WT1 peptides made WT1-specific CTLs, which were generated by MLPC, less reactive to MHC/WT1 tetramers and the unreactivity lasted at least for 2 weeks. The present study showed that WT1 peptide vaccination for an imatinib-pretreated CML patient is feasible and effective, which is due to the generation of WT1-specific CTLs with cytotoxicity against WT1-expressing leukemia cells. In addition, since the CTLs are suggested to get tolerant to WT1 peptides presented by leukemia cells by the exposure with WT1 peptides, the interval of peptide administration has to be considered for the clinical efficacy of peptide vaccination. The present findings including in vivo and in vitro studies revealed that the administration of the peptides every 4 weeks is superior to every 2 weeks. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5117 Although cellular immunotherapy based on antigen-specific cytotoxic T lymphocytes (CTLs) against tumors including leukemia and severe infections is a promising strategy, one of the pivotal issues is a hardship in constant supply of high quality antigen presenting cells for generating autologous CTLs against antigens associated with tumor cells or pathogens. We established a leukemic plasmacytoid dendritic cell (pDC) line (PMDC05) with potent antigen presenting capacity from leukemia cells of a HLA-A*0201/*2402 patient with pDC acute leukemia. We investigated whether PMDC05 could be efficiently used for generating CTLs specific for antigens of leukemia cells or pathogens and whether PMDC05 could be applicable for cellular immunotherapy for tumors and infections. PMDC05, which grew in the absence of feeder cells, was positive for CD4, CD56, CD33, HLA-DR, CD123 (IL-3Rα) and CD86 in the absence of lineage markers. mRNA of TLR1, TLR2, TLR4, TLR7 and TLR9 were clearly expressed and among these TLRs, TLR7 was prominent. Transcripts of preTα, SpiB, MX1, IRF1, IRF7 and IFN-α14 were markedly expressed in PMDC05 and those of λ-like 14.1, IL-12p35 or IL-12p40 were also expressed. By culturing PMDC05 with IL-3, CpG-B or LPS, the expression of CD1a, CD80, CD83, CD86 or HLA-DR was remarkably enhanced. Transcripts of IRF1, IRF7, IFN-α14, IL-12p35 and IL-12p40 were increased by culturing with CpG-A, CpG-B, influenza virus or LPS. Production of IFN-α and IL-12p75 was enhanced by the stimulation with CpG-A and LPS, respectively. PMDC05 possessed a considerable antigen presenting ability to naïve T cells, which was enhanced by culturing with IL-3 or influenza virus and especially by LPS, and the ability to uptake lucifer yellow. These findings revealed that PMDC05 is a unique leukemic pDC cell line with potent antigen presenting ability. Therefore, we tried to generate WT1 or CMV-specific CTLs by using PMDC05 pulsed with relevant antigen as antigen presenting cells. PMDC05, which was stimulated with 0.1 μg/ml LPS and loaded with 10 μg/mL WT1 peptides (HLA-A*2402-restricted, modified-type 9-mer peptide; CYTWNQMNL) or CMV pp65 peptides (HLA-A*2402-restricted, 9-mer peptide; QYDPVAALF) for 24 hours, was irradiated (60 Gy) and co-cultured with allogeneic CD8+ T cells purified from peripheral blood mononuclear cells (PB-MNCs) of HLA-A*2402+ healthy donor (PMDC05:CD8+ Tcells = 1:10). 50 U/ml IL-2 was added to the co-culture at day 2, and IL-2 as well as 10 ng/ml IL-7 were added every 3 days thereafter. Burkitt lymphoma cell line, Daudi, was used as control of PMDC05 in the same manner. PB-MNCs, which were presumed to contain regulatory T (Treg) cells, were also co-cultured with PMDC05 or Daudi. Induction of WT1 or CMV-specific CTLs was evaluated by flow cytometry analysis using HLA-A*2402 WT1 tetramer or CMV tetramer every week. Although Daudi pulsed with WT1 peptides could not induce WT1 tetramer+CD8+ T cells during the co-culture up to 7 weeks, PMDC05 began to induce WT1 tetramer+CD8+ T cells at week 4 and the percentage of WT1 tetramer+ T cells in CD8+ T cells rose to more than 75% at week 7. Likewise, CMV tetramer+CD8+ T cells were amplified in CD8+ T cells co-cultured with CMV peptide-pulsed PMDC05 but not in those with Daudi. Although CMV tetramer+CD8+ T cells were generated in MNCs co-cultured with PMDC05 pused with CMV peptides, WT1 tetramer+CD8+ T cells were not observed in in MNCs co-cultured with PMDC05. These data suggested that PMDC05 could be efficiently used for generating CTLs specific for antigens of tumors or pathogens and could be applicable for cellular immunotherapy for tumors and infections. In addition, it was suggested that generation of antigen-speific CTLs might be affected by co-existing Treg cells in some tumor specific antigens. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2461 Poster Board II-438 3′ trailers of pre-tRNA or micro-pre-tRNA are removed by tRNA 3′ processing endoribonuclease (tRNase ZL) for pre-tRNA or micro-pre-tRNA to transform into mature tRNA or micro-tRNA. It has been demonstrated that 7-base-pair acceptor stem, about 5-base pair T-loop structure and a 3′-trailer are requisites for being recognized by tRNase ZL. A synthesized small guide RNA (sgRNA), which is designed to possess the complementary sequences with target RNA and form sgRNA/target RNA complex resembling a pre-tRNA or a micro-pre-tRNA, could be used for target RNA to be recognized and cleaved. As to the structure of sgRNA, a 3′-truncated tRNA (a clover leaf-like structure resembling tRNA and complementary to target RNA in the 7-base-pair acceptor stem) and a 5′-half-tRNA (a half of clover leaf-like structure and complemantary to target RNA of a 3′-half-tRNA structure) can form a pre-tRNA-like complex with target RNA. Heptamer sgRNA (7-base RNA structure complementary to target RNA and making target RNA form T-loop structur downstream of the complementary region) as well as hook RNA (a single T-loop-like structure with upstream 7-base RNA structure complementary to target RNA) can form a micro-pre-tRNA-like complex, which could be recognized and cleaved by tRNase Z. Heptamer sgRNA is thought to be superior to siRNA in mucleic acid medicine because of less off-target effect, less IFN-α production, easier introduction into target cells and less expensive for synthesis. In order to develop a unique sgRNA-based nucleic acid medicine, we investigated whether apoptosis could sbe induced and proliferation could be inhibited in leukemia cells by using tumor antigen heptamer sgRNA. The 5′, 3′-phosphorylated 2′-O-methyl sgRNAs with or without 3′LNA (locked nucleic acid), which were designed to form micro-pre-tRNA like structure with target mRNA of leukemia-associated antigens (LAA) such as WT1, Bcl2, PRAME, survivin, proteinase 3, hTERT and RHAMM, were synthesized and purified by high-performance liquid chromatography. Several leukemic cell lines and fresh leukemia cells from AML/ALL patients expressing relevant LAA were cultured with each heptamer sgRNA at the concecntrations of 0.1 μM to 10 μM for 24-72 hours. Effecs of sgRNA on apoptosis of the leukemia cells were evaluated by flow cytometry after staining with Annexin-V/7AAD or DiOC6(3). Proliferaton inhibition of the cells by sgRNA was evaluated by an improved MTT assay (TetraColor One). In addition, introduction of heptamer sgRNA into target cells was investigated using FITC-labeled sgRNA. Although EGFP sgRNA (negative control) did not induce apoptosis in leukemia cells such as K562, heptamer sgRNA against WT1, Bcl2, PRAME, surviving, proteinase 3, hTERT and RHAMM could induce apoptosis in concentration dependent manner of sgRNA irrespective of sgRNA with or without LNA. Apoptosis was enhanced by the combination of two different sgRNAs such as WT1 sgRNA and PRAME sgRNA. Likewise, proliferation was inhibited in leukemia cells cultured with sgRNA against WT1, PRAME, survivin, proteinase 3, hTERT and RHAMM. Apoptosis due to anti-leukemic agent such as etoposide or doxorubicine was enhanced by the addition of sgRNA more than the additive theoretical level of anti-leukemic agent and sgRNA. Apoptosis of some leukemia cells such as HL60, which were resistant to LAA sgRNA in apoptosis or proliferation inhibition, could be hardly introduced into with FITC-labeled sgRNA by only culturing with. sgRNA-based apoptosis in these cell lines such as HL60 was enhanced by using lipofection for sgRNA intruduction. These findings suggested a possibility that tumor antigen sgRNA could be applied as a novel nucleic acid medicine against leukemia and other tumors. Disclosures: No relevant conflicts of interest to declare.