Akiko Kikuchi

Activation of human Vα24NKT cells by α-glycosylceramide in a CD1d-restricted and Vα24TCR-mediated manner

Vα14NK(natural killer) T cells play an important role in controlling tumors or in preventing autoimmunity in the murine system. Vα24NKT cells, the human counterpart of Vα14NKT cells, may contribute to controlling the progression of autoimmune diseases in humans. These findings show the possibility that ligand(s) for these NKT cells can control the above-mentioned pathological conditions. Specific glycolipids such as α-galactosylceramide (α-GalCer) and α-glucosylceramide (α-GlcCer) have been identified as ligand(s) recognized by murine Vα14NKT cells in a CD1d-restricted manner, but it remains unclear whether these glycolipids are ligand(s) for Vα24NKT cells in humans. To determine whether α-glycosylceramide is presented by CD1d molecules in humans, we initially established a Vα24NKT cell line specific for α-glycosylceramide using dendritic cell (DC) like cells from normal peripheral blood mononuclear cells (PBMC) in an autologous mixed leukocyte reaction (auto-MLR) system, and characterized the Vα24NKT cell line. The Vα24NKT cells were CD3+CD4−CD8−Vα24+Vβ11+NKRP1A+ and specifically proliferated in response to α-glycosylceramide in CD1d-restricted and Vα24TCR-mediated manner. The phenotypic and functional similarities between murine Vα14NKT cells and human Vα24NKT cells suggest that Vα24NKT cells may play an important role in controlling tumors or in preventing autoimmunity as observed with Vα14NKT cells.

α-Glycosylceramides Enhance the Antitumor Cytotoxicity of Hepatic Lymphocytes Obtained from Cancer Patients by Activating CD3−CD56+ NK Cells In Vitro

α-Glycosylceramides, such as α-galactosylceramide and α-glucosylceramide, induce antitumor immunity in various murine cancer models. In the murine hepatic metastasis model, Vα14 TCR+NK1.1+ T cells, which accumulate preferentially in the liver, are considered to play a key role in the induction of antitumor immunity by α-glycosylceramides. We recently reported that Vα24 TCR+ NKT cells, the human homologues of murine Vα14 TCR+NK1.1+cells, are rarely seen among freshly isolated human hepatic lymphocytes. Therefore, it is important to examine whether α-glycosylceramides also enhance the antitumor cytotoxicity of human hepatic lymphocytes, as they have been shown to do in murine systems, to determine the usefulness of α-glycosylceramides in cancer immunotherapy in humans. Here, we show that α-glycosylceramides greatly enhance the cytotoxicity of human hepatic lymphocytes obtained from cancer patients against the tumor cell lines, K562 and Colo201, in vitro. The direct effector cells of the elicited cytotoxicity were CD3−CD56+ NK cells. Even though Vα24 TCR+NKT cells proliferated remarkably in response to α-glycosylceramides, they did not contribute directly to the cytotoxicity. Our observations strongly suggest the potential usefulness of α-glycosylceramides for immunotherapy of liver cancer in humans based on their ability to activate CD3−CD56+ NK cells in the liver.

Human peripheral blood Vα24+ Vβ11+ NKT cells expand following administration of α-galactosylceramide-pulsed dendritic cells

Background and Objectives We have undertaken the first clinical trial involving the administration of α‐GalactosylCeramine (α‐GalCer)‐pulsed dendritic cells (DCs) to human subjects, to determine safety, optimal dose, optimal administration route and immunological effects.

Profiling of genes expressed in human monocytes and monocyte-derived dendritic cells using cDNA expression array

Using a human cDNA expression array, we obtained expression profiles of 588 genes in CD14+ monocytes and monocyte‐derived dendritic cells (DCs). Overall, 22 genes were upregulated, and nine genes were downregulated in DCs of both samples from two different individuals. Many of the genes that were upregulated in DCs encode proteins that are related to differentiation, cell structure, migration, termination of cell cycle as well as proliferation, e.g. tumour necrosis factor‐α (TNF‐α), tumour necrosis factor receptor II (TNFRII), thymosin β‐10, epithelial discoidin domain receptor 1, replication factor C, putative transcription factor DB1, alpha catenin, transforming growth factor‐β1, prohibitin, p53‐regulating protein and neu differentiation factor. Among the downregulated genes in DCs were genes that encode proteins of cell cycle regulation: mitotic growth and transcription activator, platelet‐derived growth factor receptor‐β subunit, interleukin 2 receptor (IL‐2R)‐γ subunit, IL‐7R‐α subunit, leucocyte interferon‐γ (IFN‐γ) and granulocyte–macrophage colony‐stimulating factor receptor (GM‐CSFR). Semi‐quantitative reverse transcription–polymerase chain reaction method confirmed the upregulated expression levels in DCs for TNFRII, TNF‐α, alpha catenin and downregulation of IFN‐γ, GM‐CSFR on four different donor samples of DCs and monocytes. Moreover, our data show the presence of a ‘switch‐on’ step for the TNF‐α and TNFRII gene expression in immature DCs for further differentiation into mature DCs.

Binding of nonamer peptides to three HLA-B51 molecules which differ by a single amino acid substitution in the A-pocket

The interaction between 9-mer peptides and HLA-B51 molecules was investigated by quantitative peptide binding assay using RMA-S cell expressing human β2-microglobulin and HLA-B51 molecules. Of 147 chemically synthesized 9-mer peptides possessing two anchor residues corresponding to the motif of HLA-B*5101 binding self-peptides, 27 paptides bound to HLA-B*5101 molecules. Pro and Ala at position 2 as well as Ile at position 9 were confirmed to be main anchor residues, while Gly at position 2 as well as Val, Leu, and Met at position 9 were weak anchor residues for HLA-B*5101. The A-pocket is suspected to have a critical role in peptide binding to MHC class I molecules because this pocket corresponds to the N-terminus of peptides and has a strong hydrogen bond formed by conserved Tyr residues. Further analysis of peptide binding to HLA-B*5102 and B*5103 molecules showed that a single amino acid substitution of Tyor for His at residue 171(B*5102) and that of Gly for Trp at residue 167 (B*5103) has a minimum effect in HLA-B51-peptide binding. Since previous studies showed that some HLA-B51 alloreactive CTL clones failed to kill the cells expressing HLA-B*5102 or HLA-B*5103, these results imply that the structural change of the A-pocket among HLA-B51 subtypes causes a critical conformational change of the epitope for TCR recognition rather than influences the interaction between peptides and MHC class I molecules.

The CD1d natural killer T-cell antigen presentation pathway is highly conserved between humans and rhesus macaques

Natural killer T (NKT) cells play an important role in controlling cancers, infectious diseases and autoimmune diseases. Although the rhesus macaque is a useful primate model for many human diseases such as infectious and autoimmune diseases, little is known about their NKT cells. We analyzed Vα24TCR+ T cells from rhesus macaque peripheral blood mononuclear cells stimulated with α-galactosylceramide (α-GalCer) and interleukin-2. We found that rhesus macaques possess Vα24TCR+ T cells, suggesting that recognition of α-GalCer is highly conserved between rhesus macaques and humans. The amino acid sequences of the V–J junction for the Vα24TCR of rhesus macaque and human NKT cells are highly conserved (93% similarity), and the CD1d α1–α2 domains of both species are highly homologous (95.6%). These findings indicate that the rhesus macaque is a useful primate model for understanding the contribution of NKT cells to the control of human diseases.

Residue 116 determines the C-terminal anchor residue of HLA-B*3501 and -B*5101 binding peptides but does not explain the general affinity difference

Abstract HLA-B*3501 and -B*5101 molecules, which belong to the HLA-B5 cross-reactive group, bind peptides carrying similar anchor residues at P2 and the C-terminus, but differences are observed in the preference for a Tyr residue at the C-terminus and the affinity of peptides. A recent study of HLA-B*3501 crystal structure suggested that residue 116 on the floor of the F-pocket determines a preference for anchor residues at the C-terminus. In order to evaluate the role of the residue 116 in the peptide binding to both HLA-B*3501 and HLA-B*5101 molecules, we generated HLA-B*3501 mutant molecules carrying Tyr at residue 116 (B*3501–116Y) and tested the binding of a panel of nonamer peptides to the B*3501–116Y molecules by a stabilization assay with RMA-S transfectants expressing the mutant molecules. The substitution of Tyr for Ser at residue 116 markedly reduced the affinity of nonamer peptides carrying Tyr at P9, while it enhanced that of nonamer peptides carrying Ile and Leu at P9. On the other hand, the affinity of peptides carrying aliphatic hydrophobic residues at P9 to B*3501–116Y molecules was much higher than that to HLA-B*3501 and HLA-B*5101 molecules. These results indicate that residue 116 is critical for the structural difference of the F-pocket between HLA-B*3501 and HLA-B*5101 which determines the C-terminal anchor residues, while leaving other residues which differ between HLA-B*3501 and HLA-B*5101 may be responsible for the low peptide binding property of the latter.

Identification of the gene encoding a novel HLA-B39 subtype Two amino acid substitutions on the β-sheet out of the peptide-binding floor form a novel serological epitope

Serological analysis suggests the existence of a novel HLA-B39 subtype (HLA-B39N) in the Japanese population. To identify this novel allele, a gene encoding HLA-B39N was cloned and the exons were sequenced. A gene encoding HLA-B39N (B*3904) and B*39011 differs by two nucleotide substitutions at codons 11 and 12 whereas B*3904 and B*39013 differ by three nucleotide substitutions at codons 11, 12, and 312. One nucleotide difference at codon 11 produces a change from serine in B*3901 to alanine in B*3904 whereas another difference at codon 12 changes valine in B*3901 to methionine in B*3904. The residues 11 and 12 are located on the beta-sheet out of the peptide-binding floor and are completely buried in the molecule. These results suggest that the substitutions at these residues alter the conformation of other residues forming epitopes of alloantibodies. Analysis of HLA-B*3901 genes in the Japanese population showed that both B*39011 and B*39013 were observed in the Japanese population. The present study suggests that B*3904 may have evolved from B*39011 rather than B*39013.

Experience-dependent Plasticity of the Optomotor Response in Drosophila melanogaster

Experience in early life can affect the development of the nervous system. There is now evidence that experience-dependent plasticity exists in adult insects. To uncover the molecular basis of plasticity, an invertebrate model, such as Drosophila melanogaster, is a powerful tool, as many established genetic and molecular methods can be applied. To establish a model system in which behavioral plasticity can be examined, we investigated the optomotor response, a behavior common to most sight-reliant animals, in Drosophila and found that the response could be modified by the level of light during rearing. The angle turned by the head in response to a moving stimulus was used to quantify the response. Deprivation of light increased the response to low-contrast stimuli in wild-type Drosophila at 4 days after eclosion and this plastic change did not appear in rutabaga, a known mutant defective in short-term memory. In addition, the change was transient and was markedly decreased at 6 days after eclosion. Further, we found that Dark-flies, which have been kept in constant darkness for more than 50 years, showed a higher response to low-contrast stimuli even at 6 days after eclosion compared to wild type and this characteristic was not lost in Dark-flies placed in a normal light environment for 2 generations, suggesting that this high response has a hereditary nature. Thus, our model system can be used to examine how the environment affects behaviors.

Influence of Al Composition Thickness on Critical Current Density in Transformation-Processed

The influence of the Al layer thickness on the critical current density Jc of transformation-processed Nb3Al superconductors was investigated. The Al layer thickness in the jerry-roll filaments of precursor wires was varied from 120 to 250 nm; the Nb layer thickness is proportional to the Al thickness, keeping the Nb-Al composition ratio to 3. The rapid heating and quenching (RHQ) operation condition was varied within a range of the conditions to form the ductile body-centered cubic (BCC) Nb-Al phase. The RHQ wires were also followed by an areal reduction process prior to the Nb3Al phase transformation. Thinner Al thickness wires show a steeper increase in Jc with reduction in the area, whereas thicker Al thickness wires show a slower increase. The best Jc performance was obtained for the thinnest Al thickness sample. Without areal reduction, there was not much difference in the optimum Jc with respect to the Al thickness.