Search

Purpose: The interleukin-13 receptor alpha 2 (IL-13R alpha 2) is a glioma-restricted cell-surface epitope not otherwise detected within the central nervous system. Here, we report a novel approach for targeting malignant glioma with IL-13R alpha 2-specific CTLs., Experimental Design: Artificial antigen-presenting cells (aAPC) were made by coating human leukocyte antigen (HLA)-A2/pIL-13R alpha 2(345-354) tetrameric complexes, anti-CD28 antibody, and CD83 molecules to cell-sized latex beads, and used to stimulate IL-13R alpha 2-specific CTLs from the peripheral blood mononuclear cells of HLA-A2+ healthy donors. After multiple stimulations, the induced CTLs were analyzed for tetramer staining, IFN-gamma production, and CTL reactivity., Results: Tetramer staining assay showed that the induced CTLs specifically bound HLA-A2/pIL-13R alpha 2(345-354) tetramers. The CTLs specifically produced IFN-gamma in response to the HLA-A2/pIL-13R alpha 2(345-354)-aAPCs and exhibited specific lysis against T2 cells pulsed with the peptide pIL-13R alpha 2(345-354) and HLA-A2+ glioma cells expressing IL-13R alpha 2(345-354), whereas HLA-A2(-) glioma cell lines that express IL-13R alpha 2(345-354) could not be recognized by the CTLs. The peptide-specific activity was inhibited by anti-HLA class I monoclonal antibody., Conclusion: The induced CTLs specific for IL-13R alpha 2(345-354) peptide could be a potential target of specific immunotherapy for HLA-A2+ patients with malignant glioma.

Interleukin-13 receptor alpha2 (IL-13Ralpha2) is a glioma-restricted cell-surface epitope not otherwise detected within the central nervous system. The present study is a report of a novel approach of targeting malignant glioma with IL-13Ralpha2-specific cytotoxic T lymphocyte (CTL) induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with human leukocyte antigen (HLA)-A2-restricted IL-13Ralpha2(345-353) peptide-pulsed T2 cells. The induced CTL showed specific lysis against T2 cells pulsed with the peptide and HLA-A2+ glioma cells expressing IL-13R2(345-353), while HLA-A2 glioma cell lines that express IL-13Ralpha2(345-353) could not be recognized by CTL. The peptide-specific activity was inhibited by anti-HLA class I monoclonal antibody. These results suggest that the induced CTL specific for IL-13Ralpha2(345-353) peptide could be a potential target of specific immunotherapy for HLA-A2 patients with malignant glioma.

The role of the bound peptide in alloreactive T-cell recognition is controversial, ranging from peptide-independent to peptide-specific recognition of alloreactive T-cells. The aim of this study is to find the evidence that there exist peptide/MHC complex (pMHC)-specific CTLs among alloreactive T cells generated with long-term mixed lymphocytes culture (LTMLC). A single pMHC was manipulated by loading the TAP-defective, HLA-A2 expressing T2 cells with a viral peptide (LMP2A(426-434)) or a self-peptide (Tyr(369-377)). The PBLs samples from 4 HLA-A2 positive (HLA-A2+ve) and 4 HLA-A2 negative (HLA-A2-ve) donors were included in this study. The HLA-A2+ve PBL co-cultured with the LMP2A(426-434) pulsed T2 (T2/LMP) stands for the nominal T-cell response to a viral antigen, and the HLA-A2-ve PBLs co-cultured with the Tyr(369-377) pulsed T2 (T2/Tyr) for alloreactive T-cell response to an allogeneic antigen. The specificity of the expanded CTLs after the LTMLC was detected by their specific cytotoxicity and binding ability to specific pMHC-tetramer. An HLA-A2 restricted, HIV peptide (Gag(77-85)) was included for control. The cultural bulk of HLA-A2+ve PBLs with the T2/LMP showed an elevated specific cytotoxicity against the T2/LMP compared to that against the T2/HIV (26.52%+/-3.72% vs 7.01%+/-0.87%, P<0.001), and an increased frequency of binding to LMP-tetramer compared to that binding to HIV-tetramer (0.98%+/-0.33% vs 0.05%+/-0.01%, P=0.0014). The cultural bulk of HLA-A2-ve PBLs with the T2/Tyr showed a more active cytotoxicity against the T2/Tyr than that against T2/HIV (28.07%+/-2.58% vs 6.87%+/-1.01%, P<0.001), and a higher frequency of binding to the Tyr-tetramer than that binding to the HIV-tetramer (0.88%+/-0.3% vs 0.06%+/-0.03%, P=0.0018). Our results indicate that the LTMLC is able to expand the viral antigen-specific CTLs as well as allogeneic antigen-specific CTLs. A relatively large proportion of alloreactive CTLs should be pMHC-specific, i.e., the specificity of the alloreactive lines depends on both the bound peptide and the allotype of MHC. Our observations support the hypothesis that the cumulative effect of T cells specific to each peptide epitope could account for the strength and diversity of the alloresponse. The method using manipulated pMHC and the LTMLC to generate pMHC-specific, alloreactive CTLs is of potential importance for adoptive T-cell immunotherapy.

HLA-A2 is the most frequent HLA-A allele in all ethnic populations, and an important restriction element for peptide presentation to T cells in infectious disease and cancer. However, the HLA-A2 supertype consisting of up to 75 subtypes, mutation studies and analyses using cytotoxic T lymphocytes suggest the functional relevance of subtype-specific differences in HLA-A2 molecules for peptide binding and T-cell recognition. Therefore, it is necessary for T-cell response study to discriminate the HLA-A2 subtypes and to understand the profile of HLA-A2 allelic distribution in a given population. In this study, we developed a simple, robust approach based on the nested polymerase chain reaction using sequence-specific primers (PCR-SSP) to discriminate 17 HLA-A2 subtypes which cover the most HLA-A2 alleles (> 99% allele frequency) reported in Chinese, using 15 combinations of 19 allelic specific primers. In the first round of PCR, 3 combinations of 5 primers were used to determine whether the tested sample was HLA-A2 positive, meanwhile the subtypes of HLA-A*0209 and HLA-A*0215N were determined for the variant position of these two subtypes is in exon 4 instead of exon 2, 3. Samples of HLA-A2 positive were subtyped in the second round of PCR, using PCR products of the first round as templates. This strategy was applied to test the samples of 78 random HLA-A2 positive individuals for their HLA-A2 subtypes. Those samples were screened for HLA-A2 positive by the first round PCR-SSP from 154 healthy blood donors in Wuhan, China. The subtyping results were verified by using flow cytometric analysis (FCM) with HLA-A2 specific monoclonal antibody BB7.2 and DNA sequencing. The typing results of the samples show 50.7% random individuals in the population carry HLA-A2, HLA-A*0201 ranks the first (allele frequency = 15.5%), followed by A*0207 (5.8%), A*0206 (4.7%), A*0203 (2.6%), A*0210 (0.7%), and these 5 alleles account for 99.0% HLA-A2 subtypes of allele frequency. Our study indicates that the developed typing method is simple and reliable for HLA-A2 subtyping in Chinese, and the profile of allelic distribution of HLA-A2 subtypes is revealed in the population of Wuhan, China.

An efficient, six-step stereocontrolled total synthesis of the antifungal agent strobilurin B is reported and was based on a convergent bi-directional coupling featuring a hetero-bis-1,4-metallated pentadiene system as the linchpin.

HLA-A*2402 is one of the most frequent HLA-A allele in Asian population. To construct HLA-A*2402-peptide tetramers, the transmembrane and intracellular segments of HLA-A*2402 cDNA were replaced with BSP sequence to form a fusion gene of sHLA-A*2402-BSP. The sHLA-A*2402-BSP fusion protein and beta2m were high-level expressed as insoluble aggregates in E.coli, and refolded to form an HLA-A*2402-peptide monomeric complex by dilution method in the presence of an antigenic peptide. The HLA-A*2402-peptide monomeric complex was biotinated and tetramized to prepare HLA-A*2402-peptide tetramer. Then using the HLA-A*2402-peptide tetramers to detect antigen-specific cytotoxic T lymphocyte (CTL) induced by artificial antigen presenting cell (aAPC) in vitro. The results showed that HLA-A*2402-peptide tetramer was prepared correctly, and functional in detecting antigen-specific CTL in vitro, HLA-A*2402-peptide monomeric and its multimeric complexes are expected to provide a powerful tool for studying mechanisms of immune-related diseases in Asian populations.

The HOXA9 homeodomain protein is a key regulator of hematopoiesis and embryonic development. HOXA9 is expressed in primitive hematopoietic cells, and its prompt downregulation is associated with myelocytic maturation. Although transcriptional inactivation of HOXA9 during hematopoietic differentiation has been established, little is known about the biochemical mechanisms underlying the subsequent removal of HOXA9 protein. Here we report that the CUL-4A ubiquitylation machinery controls the stability of HOXA9 by promoting its ubiquitylation and proteasome-dependent degradation. The homeodomain of HOXA9 is responsible for CUL-4A-mediated degradation. Interfering CUL-4A biosynthesis by ectopic expression or by RNA-mediated interference resulted in alterations of the steady-state levels of HOXA9, mirrored by impairment of the ability of 32D myeloid progenitor cells to undergo proper terminal differentiation into granulocytes. These results revealed a novel regulatory mechanism of hematopoiesis by ubiquitin-dependent proteolysis.

[reaction: see text] 1,3-Prototropic rearrangement of the benzannulated enyne-isonitriles to the corresponding enallene-isonitriles followed by cycloaromatization generated the putative quinoline biradicals/zwitterions. A subsequent intramolecular radical-radical coupling or electrophilic aromatic substitution then gave the formal [4 + 1] cycloaddition adducts leading to 11H-indeno[1,2-b]quinoline and related compounds.

Several derivatives of the pyrido[1',2':1,2]pyrimido[4,5-b]indoles 4 and the pyrazino[1',2':1,2]pyrimido[4,5-b]indoles 14 were synthesized by treatment of the benzannulated enyne-isocyanates 8 with the iminophosphoranes 9 and 13, respectively, for the aza-Wittig reaction followed by thermolysis. The reaction presumably proceeds through an initial formation of the corresponding benzannulated enyne-carbodiimides, such as 10, followed by a formal intramolecular hetero Diels-Alder reaction. Surprisingly, when the iminophosphorane 17 was used for condensation with 8, the expected pyrimido[1',6':1,2]pyrimido[4,5-b]indoles 16 were not obtained. Instead, the isomeric pyrimido[6',1':2,3]pyrimido[4,5-b]indoles 21 were isolated. Presumably, an alternative reaction pathway involving an initial [2 + 2] cycloaddition reaction to form 19 followed by ring opening could lead to 20 and, after an intramolecular radical-radical coupling, 21. Treatment of the urea derivatives 24 with dibromotriphenylphosphorane also produced in situ the benzannulated enyne-carbodiimides 25, which on thermolysis gave the isoquinolino[2',1':1,2]pyrimido[4,5-b]indoles 26. Methylation of 4a, 14a, and 26a with methyl iodide occurred exclusively at the site of the indolo nitrogen. The planar geometry of those novel heteroaromatic compounds, resembling many DNA-binding agents, makes them potential candidates as DNA intercalators.

New synthetic pathways to the 5H-pyrido[3',4':4,5]pyrrolo[2,3-b]quinolines 6, the 6H-pyrido[3',4':4,5]pyrrolo[2,3-b][1,6]naphthyridines 7, and the 11H-pyrido[4',3':4,5]pyrrolo[2,3-b]quinolines 8 via thermolysis of the pyridannulated enyne-carbodiimides 14, 19, and 23 were established. These novel heteroaromatic systems are structurally related to ellipticine alkaloids and could serve as DNA-intercalating agents.