マイナー組織適合抗原高親和性T細胞レセプター遺伝子導入による特異的CTLの誘導

本研究は、接着分子L-セレクチンの多型部位を含む新規マイナー組織適合抗原ペプチドに特異的かつ増殖活性の高い細胞傷害性T細胞を誘導し、難治性白血病治療に用いることを最終目標としている。最初に、HLA一致ドナーからの同種造血幹細胞移植が予定されているHLA-A*2402陽性白血病患者およびドナーのL-セレクチンアレルの同種多型を、PCR-RFLP法により決定した。HLA-A*2402を保有し、患者のL-セレクチンがS/S型またはP/S型、ドナーのL-セレクチンがP型の白血病患者由来の白血病細胞および末梢血・ドナー由来の末梢血を採取し保存した。患者およびドナーから採取した末梢血単核細胞を、EBウイルス産生株B95-8の上清を加えて培養することにより、lymphoblastoid cell line(LCL)を作成した。同時に患者の骨髄を液体培養することにより線維芽細胞も作成した。次に、HLA-A*2402分子への親和性を考慮し、L-セレクチン多型部位を含む9アミノ酸(P型,S型各9種類)を全て合成した。IFNγ産生能を指標に候補ペプチドをスクリーニングし、HLA stabilization assayを利用して、ペプチドとHLA-A*2402分子との結合性をフローサイトメトリーで確認した。さらに、L-セレクチン多型部位を含む100bpの遺伝子とHLA-A*2402遺伝子を導入したT2-A24/L-selectin細胞に対する細胞傷害活性誘導能を指標に、S2ペプチドがマイナー組織適合抗原として機能することを確認した。S2ペプチド特異的細胞傷害性T細胞を、S2ペプチド/HLA-A24テトラマーと反応させ、PE蛍光色素の強さをフローサイトメトリーで解析し、T細胞レセプターとS2ペプチドとの結合力を評価した。In hematopoietic SCT from HLA-matched donors, allogeneic immune reactions such as GVHD and GVL effect are induced by disparities in minor histocompatibility antigens (mHas) between donor and recipient. CD62L has been reported to act as an mHa based on analysis of the outcome of allogeneic SCT. To identify peptides derived from CD62L that may be involved in GVL effect, we prepared all possible 18 different 9-mer peptides derived from CD62L that include codon 213 in one of the 9 positions, and tested for cytotoxic T-cell precursors specific to any of these peptides in patients after CD62L-disparate SCT. PBMC obtained from 5 patients with I-ILA-A*2402 who developed GVHD following transplantation were incubated with autologous monocyte-derived DCs or HLA-A*2402-transfected T2 cells which were pulsed with the 9-mer peptides, or incubated with the peptides only, and 1FN-gamma secretion from PBMC after 16 hours incubation was examined. Two to 4 different recipient-type peptides for each patient induced higher IFN-gamma secretion than the others. By stimulating PBMC with the peptide-pulsed T2-A24 cells, T-cell lines specific for 4 peptides were obtained. The cultured T cells efficiently lysed peptide-pulsed T2-A24 cells as well as HLA-A*2402-positive lymphoblastic cell line cells that were disparate with CD62L. Addition of mAbs against HLA-class I blocked this cytotoxicity. WIC stabilization assay demonstrated that the 4 peptides bound to the cell surface and HLA complex stabilized. These results suggest these peptides may serve as mHas capable of inducing GVL effect, and CTLs recognizing CD62L-derived polymorphic peptides may have therapeutic value in treating relapsed leukemia after SCT.研究課題/領域番号:18591049, 研究期間(年度):2006 – 2007出典：研究課題「マイナー組織適合抗原高親和性T細胞レセプター遺伝子導入による特異的CTLの誘導」課題番号18591049（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-18591049/185910492007kenkyu_seika_hokoku_gaiyo/）を加工して作

再生不良性貧血患者末梢血単核球における熱ショック蛋白質72の誘導性亢進: シクロスポリン療法が効きやすい免疫性再生不良性貧血のマーカーとしての意義

取得学位：博士(医学), 学位授与番号：医博乙第1518号，学位授与年月日：平成12年3月1日,学位授与年：200

INFγ発現とT細胞レセプターを指標としたマイナー組織適合抗原特異的T細胞の検出

金沢大学医薬保健研究域医学系Y染色体特異的マイナー組織適合抗原であるH-Yペプチドは男性にのみ発現し,HLA-A2に表出される.まず,HLA一致姉からの同種骨髄移植後に慢性骨髄性白血病リンパ性急性転化を来した男性患者(HLA-A2陽性)を対象に,患者の白血病細胞に対し高い細胞傷害活性を示すドナー由来のT細胞を作成した.H-Yペプチドでパルスしたドナー由来樹状細胞を抗原提示細胞に用いてドナーのT細胞を繰り返し刺激することにより,細胞傷害性T細胞を樹立した.この細胞傷害性T細胞は,ドナーの樹状細胞に対しパルスするペプチド濃度依存性の細胞傷害活性を示した.さらに,患者白血病細胞・線維芽細胞,男性リンパ芽球性リンパ細胞株細胞に対し,HLA-A2拘束性の細胞傷害活性を示した.次に,同じ男性患者が再移植を受けたのち分子遺伝学的寛解を維持していた時期の流血中に,インターフェロン(IFN)γ発現とT細胞レセプターを指標としてH-Yペプチド特異的T細胞を検出した.患者血液からCD8陽性細胞を分離し,H-Yペプチドで5時間刺激したところ,6.8%がIFN-γ陽性となった.このCD8陽性IFN-γ陽性細胞のT細胞レセプターβ鎖のCDR3領域をPCRにより増幅し,サイズ・スペクトラタイピングを行った.その結果,BV22においてin vitroで誘導された細胞傷害性T細胞と同じサイズのピークが検出された.塩基配列を決定したところ,患者の流血中に存在しているH-Yペプチド反応性T細胞とin vitroで樹立した細胞傷害性T細胞で共通のCDR3領域の配列が証明された.流血中に存在していたCD8陽性細胞は,H-Yペプチド特異的にIFN-γを発現することから,同種免疫による抗腫瘍効果の担当細胞であった可能性がある.研究課題/領域番号:13770578, 研究期間(年度):2001-2002出典：「INFγ発現とT細胞レセプターを指標としたマイナー組織適合抗原特異的T細胞の検出」研究成果報告書　課題番号13770578（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （ https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-13770578/ ）を加工して作

Immune pathophysiology and clinical perspective in hematopoietic stem cell transplantation

研究紹

Existence of the Wigner function with correct marginal distributions along tilted lines on a lattice

In order to determine the Wigner function uniquely, we introduce a new condition which ensures that the Wigner function has correct marginal distributions along tilted lines. For a system in $N$ dimensional Hilbert space, whose "phase space" is a lattice with $N^2$ sites, we get different results depending on whether $N$ is odd or even. Under the new condition, the Wigner function is determined if $N$ is an odd number, but it does not exist if $N$ is even.Comment: 18 page

Treatment of primary central nervous system lymphoma with induction of complement-dependent cytotoxicity by intraventricular administration of autologous-serum-supplemented rituximab

医薬保健研究域医学系We describe an immunocompetent 19-year-old man with CD20-positive primary central nervous system (CNS) lymphoma refractory to chemotherapy and irradiation. After intraventricular administration of rituximab, a chimeric anti-CD20 monoclonal antibody, supplemented with autologous serum, a remarkable response developed to the CNS parenchymal lymphoma. Cytotoxicity assays showed that untreated patient\u27s serum with rituximab, but not that of heat-inactivated patient\u27s serum with rituximab or rituximab alone, induced potent rituximab-mediated cytotoxicity against tumor cells in the patient\u27s cerebrospinal fluid, suggesting induction of complement-dependent cytotoxicity against CNS lymphoma. © 2006 Japanese Cancer Association

Macrophage colony-stimulating factor enhances rituximab-dependent cellular cytotoxicity by monocytes

医薬保健研究域医学系Recent studies suggest that monocytes are the dominant effectors by which rituximab induces cell death in B-cell lymphoma. Because macrophage colony-stimulating factor (M-CSF) can enhance the cytotoxicity of monocytes, the authors examined whether this growth factor can enhance their ability to kill lymphoma cells in vitro. Monocytes derived from a healthy volunteer were cultured for 48 h in the presence or absence of M-CSF. Monocytes stimul ated with M-CSF were significantly more cytotoxic to Daudi B-cell lymphomas than unstimulated monocytes. Flow cytometry revealed that M-CSF increased monocyte expression of Fcγ receptors III and I by 1.6- and 1.5-fold, whereas the expression of Fcγ receptor II remained unchanged. These results suggest that pretreatment with M-CSF can improve the therapeutic efficacy of rituximab against intractable CD20+ lymphoma. © 2007 Japanese Cancer Association

Ataxia-telangiectasia mutated kinase-mediated upregulation of NKG2D ligands on leukemia cells by resveratrol results in enhanced natural killer cell susceptibility

医薬保健研究域医学系The powerful activating receptor NKG2D is expressed by natural killer (NK) cells and promotes cytotoxic lysis of cancer cells expressing NKG2D ligands (NKG2D-Ls). We report the effective induction of NKG2D-Ls, achieved with the naturally occurring polyphenol resveratrol, in a broad range of leukemia cells. In this study, resveratrol upregulated the NKG2D-Ls MHC class I chain-related proteins MICA and MICB, and UL16-binding proteins ULBP1, ULBP2, and ULBP3 in most of the leukemia cells analyzed. Ligand upregulation induced by resveratrol was impaired by pharmacological and genetic disruption of ataxia-telangiectasia mutated kinase, the main regulator of NKG2D-L expression. Leukemia cells treated with resveratrol were more susceptible to killing by NK cells than untreated cells, and the enhanced cytotoxicity of NK cells was blocked by treatment of NK cells with anti-NKG2D mAbs. Interestingly, resveratrol consistently upregulated the NKG2D receptor expression and enhanced NKG2D-mediated functions in resting NK cells obtained from healthy individuals. Therefore, resveratrol has attractive immunotherapeutic potential. © 2013 Japanese Cancer Association

A Genetic Variant in the IL-17 Promoter Is Functionally Associated with Acute Graft-Versus-Host Disease after Unrelated Bone Marrow Transplantation

Interleukin IL-17 is a proinflammatory cytokine that has been implicated in the pathogenesis of various autoimmune diseases. The single nucleotide polymorphism (SNP), rs2275913, in the promoter region of the IL-17 gene is associated with susceptibility to ulcerative colitis. When we examined the impact of rs2275913 in a cohort consisting of 438 pairs of patients and their unrelated donors transplanted through the Japan Marrow Donor Program, the donor IL-17 197A allele was found to be associated with a higher risk of acute graft-versus-host disease (GVHD; hazard ratio [HR], 1.46; 95% confidence interval [CI], 1.00 to 2.13; P = 0.05). Next, we investigated the functional relevance of the rs2275913 SNP. In vitro stimulated T cells from healthy individuals possessing the 197A allele produced significantly more IL-17 than those without the 197A allele. In a gene reporter assay, the 197A allele construct induced higher luciferase activity than the 197G allele, and the difference was higher in the presence of T cell receptor activation and was abrogated by cyclosporine treatment. Moreover, the 197A allele displayed a higher affinity for the nuclear factor activated T cells (NFAT), a critical transcription factor involved in IL-17 regulation. These findings substantiate the functional relevance of the rs2275913 polymorphism and indicate that the higher IL-17 secretion by individuals with the 197A allele likely accounts for their increased risk for acute GVHD and certain autoimmune diseases