Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell neoplasm with dismal prognosis, and no optimal therapy has been developed. We tested the defucosylated chimeric anti-CC chemokine receptor 4 (CCR4) monoclonal antibody, KM2760, to develop a novel immunotherapy for this refractory tumor. In the presence of peripheral blood mononuclear cells (PBMCs) from healthy adult donors, KM2760 induced CCR4-specific antibody-dependent cellular cytotoxicity (ADCC) against CCR4-positive ATLL cell lines and primary tumor cells obtained from ATLL patients. We next examined the KM2760-induced ADCC against primary ATLL cells in an autologous setting. Antibody-dependent cellular cytotoxicity mediated by autologous effector cells was generally lower than that mediated by allogeneic control effector cells. However, a robust ADCC activity was induced in some cases, which was comparable with that mediated by allogeneic effector cells. It suggests that the ATLL patients' PBMCs retain substantial ADCC-effector function, although the optimal conditions for maximal effect have not yet been determined. In addition, we also found a high expression of FoxP3 mRNA and protein, a hallmark of regulatory T cells, in ATLL cells, indicating the possibility that ATLL cells originated from regulatory T cells. KM2760 reduced FoxP3 mRNA expression in normal PBMCs along with CCR4 mRNA by lysis of CCR4 ؉ T cells in vitro. Our data suggest not only that the CCR4 molecule could be a suitable target for the novel antibody-based therapy for patients with ATLL but also that KM2760 may induce effective tumor immunity by reducing the number of regulatory T cells.

We report the identification of two novel minor histocompatibility antigens (mHAgs), encoded by two separate single nucleotide polymorphisms on a single gene, BCL2A1, and restricted by human histocompatibility leukocyte antigen (HLA)-A*2402 (the most common HLA-A allele in Japanese) and B*4403, respectively. Two cytotoxic T lymphocyte (CTL) clones specific for these mHAgs were first isolated from two distinct recipients after hematopoietic cell transplantation. Both clones lyse only normal and malignant cells within the hematopoietic lineage. To localize the gene encoding the mHAgs, two-point linkage analysis was performed on the CTL lytic patterns of restricting HLA-transfected B lymphoblastoid cell lines obtained from Centre d'Etude du Polymorphisme Humain. Both CTL clones showed a completely identical lytic pattern for 4 pedigrees and the gene was localized within a 3.6-cM interval of 15q24.3–25.1 region that encodes at least 46 genes. Of those, only BCL2A1 has been reported to be expressed in hematopoietic cells and possess three nonsynonymous nucleotide changes. Minigene transfection and epitope reconstitution assays with synthetic peptides identified both HLA-A*2402– and B*4403-restricted mHAg epitopes to be encoded by distinct polymorphisms within BCL2A1.

Pemetrexed (MTA) is a multitargeted antifolate with promising clinical activity in lung cancer. We exposed the small cell lung cancer cell line PC6 to stepwise-increasing pemetrexed concentrations of 0.4, 1.6, and 4.0 lM, and established three pemetrexed-resistant lung cancer cell lines: PC6 ⁄ MTA-0.4, PC6 ⁄ MTA-1.6, and PC6 ⁄ MTA-4.0 cells. To investigate the mechanisms of acquired resistance to pemetrexed, we measured the expression levels of the thymidylate synthase (TS), reduced folate carrier (RFC), and folylpolygamma-glutamate synthetase (FPGS) genes. TS gene expression was significantly increased in PC6 ⁄ MTA-1.6 and PC6 ⁄ MTA-4.0 cells relative to parental cells in a pemetrexed dose-dependent manner. In contrast, the levels of RFC gene expression in PC6 ⁄ MTA-0.4 cells and FPGS in PC6 ⁄ MTA-1.6 cells were significantly decreased, whereas the levels of both genes were restored in PC6 ⁄ MTA-4.0 cells. Knockdown of TS expression using siRNA enhanced pemetrexed cytotoxicity in PC6 ⁄ MTA-4.0 cells. The expression level of the TS gene was significantly correlated with the concentration of pemetrexed for 50% cell survival (IC 50 ) in 11 non-small cell lung cancer cell lines. These results suggest that the alteration of molecular pharmacological factors in relation with pemetrexed resistance is dose-dependent, and that up-regulation of the expression of the TS gene may have an important role in the acquired resistance to pemetrexed. In addition, TS may be a predictive marker for pemetrexed sensitivity in lung cancer. (Cancer Sci 2010; 101: 161-166) P emetrexed is an MTA that targets the folate-dependent enzymes TS, DHFR, GARFT, and AICARFT, all of which are involved in the de novo biosynthesis of thymidine and purine nucleotides.(1) Pemetrexed is transported intracellularly, predominantly via the RFC, where it is metabolized to polyglutamated forms. Pemetrexed was found to be one of the best substrates for mammalian FPGS, and it is believed that polyglutamation and the polyglutamated metabolites play important roles in determining both the selectivity and antitumor activity of this agent.(2,3) The polyglutamated metabolites of pemetrexed are most active against TS, followed by DHFR, GARFT and AI-CARFT, and natural folate competes with this inhibition in all cases.(4) Therefore, the primary mechanism of the action of pemetrexed is inhibition of TS, which results in a decrease in the available thymidine necessary for DNA synthesis. (2,4) Pemetrexed is a single agent that is currently approved for second-line treatment of advanced-stage NSCLC.(5,6) In chemotherapy-naïve patients with advanced NSCLC, double combinations of platinum compounds with gemcitabine, vinorelbine, paclitaxel, and docetaxel are standard regimens. A recent phase III trial found that cisplatin ⁄ pemetrexed provides equivalent efficacy with significantly fewer side effects and more convenient administration than cisplatin ⁄ gemcitabine in advanced NSCLC.(7) Preclinical examinations found that the combination of pemetrexed with gemcitabine or...

Here we report the identification of a novel human leukocyte antigen (HLA)-B44-restricted minor histocompatibility antigen (mHA) with expression limited to hematopoietic cells. cDNA expression cloning studies demonstrated that the cytotoxic T lymphocyte (CTL) epitope of interest was encoded by a novel allelic splice variant of HMSD, hereafter designated as HMSD-v. The immunogenicity of the epitope was generated by differential protein expression due to alternative splicing, which was completely controlled by 1 intronic single-nucleotide polymorphism located in the consensus 5 splice site adjacent to an exon. Both HMSD-v and HMSD transcripts were selectively expressed at higher levels in mature dendritic cells and primary leukemia cells, especially those of myeloid lineage. Engraftment of mHA ؉ myeloid leukemia stem cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID)/␥c null mice was completely inhibited by in vitro preincubation with the mHA-specific CTL clone, suggesting that this mHA is expressed on leukemic stem cells. The patient from whom the CTL clone was isolated demonstrated a significant increase of the mHA-specific T cells in posttransplantation peripheral blood, whereas mHA-specific T cells were undetectable in pretransplantation peripheral blood and in peripheral blood from his donor. These findings suggest that the HMSD-v-encoded mHA (designated ACC - IntroductionMinor histocompatibility antigens (mHAs) are major histocompatibility complex (MHC)-bound peptides derived from cellular proteins encoded by polymorphic genes. Following human leukocyte antigen (HLA)-matched allogeneic hematopoietic cell transplantation (HCT), donor-recipient disparities in mHAs can induce a favorable graft-versus-leukemia (GVL) effect that is often associated with graft-versus-host disease (GVHD). 1-3 Significant efforts have been made to identify mHAs, particularly those specific for hematopoietic cells, since such mHAs are speculated to contribute to the GVL effect. The first report on the identification of a hematopoietic lineage-specific mHA, HA-1, was generated by the Goulmy group in 1998 (den Haan et al 4 ) as a result of biochemical analysis of peptides eluted from HLA-A*0201 molecules. The only other mHAs with selective expression in hematopoietic cells described to date are HA-2 5 ; ACC-1 and ACC-2 6 ; and DRN-7, 7 HB-1, 8,9 and PANE1, 10 the latter 2 of which are B-cell lineage-specific. Thus, identification of more mHAs should facilitate a better understanding of the biology of GVL and the development of effective immunotherapy to induce GVL reactions.Immunogenicity of most autosomal mHAs identified to date results from single-nucleotide polymorphisms (SNPs) that cause amino-acid substitutions within epitopes, leading to the differential display/recognition of peptides between HCT donor and recipient via several mechanisms: peptide binding to MHC observed in HA-1/A2-, 4 HA-2-, 5 and CTSH-encoded mHAs 11 ; proteasomal cleavage in HA-3 12 ; peptide transport in HA-8 13 ; and altered recognition o...

We examined the expression levels of the multidrug resistance protein 5 (ABCC5) gene in non -small cell lung cancer (NSCLC) cell lines to clarify the relationship with the sensitivity to gemcitabine. The expression levels of ABCC5 were inversely correlated with gemcitabine sensitivity significantly (r = 0.628; P < 0.01) in 17 NSCLC cells, whereas the expression of ABCC5 in the gemcitabine-resistant NSCLC cell line H23/GEM-R was the same as that in parental NCI-H23 cells. Treatment with the ABCC5 inhibitor zaprinast altered the sensitivity to gemcitabine in ABCC5-expressing NSCLC cells. In addition, decreasing the expression of ABCC5 by small interfering RNA altered the cytotoxicity to gemcitabine. These results indicate that modulation of ABCC5 activity could be used to increase the gemcitabine sensitivity in NSCLC. Previously, we found a decreased expression of deoxycytidine kinase in H23/GEM-R cells, and further investigation in this study showed an increased expression of ribonucleotide reductase subunit 1 in H23/GEM-R cells. We therefore also examined the effect of modifying the expression of both genes on gemcitabine resistance. We found that using small interfering RNA to decrease the expression of ribonucleotide reductase subunit 1 resulted in a decreased resistance to gemcitabine in H23/GEM-R cells. Furthermore, pretreatment with pemetrexed resulted in an increased deoxycytidine kinase expression concomitant with the alteration of the resistance to gemcitabine in H23/GEM-R cells. The determinants for sensitivity and the acquired resistance in gemcitabine are quite different; nonetheless, modification of these factors may increase the efficacy of gemcitabine in the treatment of NSCLC.