JP2008538499A - CD4T細胞の活性化方法{MethodForActivatingCD4TCells} - Google Patents
CD4T細胞の活性化方法{MethodForActivatingCD4TCells} Download PDFInfo
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Abstract
【選択図】図1
Description
T細胞を分離する方法は、特別に制限されない。例えば、細胞密度(cell density)、細胞表面エピトープに対する抗体親和度、細胞の大きさ(cell size)、蛍光放出(fluorescent emission)程度に依存して分離することができる。具体的に、アルブミン(albumin)、デキストラン(dextran)、フィコール(Ficoll)、メトリザミド(metrizamid)、 パーコール (Percoll)などを用いる密度勾配遠心分離(density gradient centrifugation)方法が、抗体を用いる方法には、MACS(magnetic activated cell sorter)などの方法が、細胞の大きさを用いる方法には、centrifugal elutriationなどの方法が、蛍光を用いる方法には、FACS等の方法がある。本発明では、単核細胞からCD4 T細胞だけを分離するために、抗-CD4 T細胞抗体を用いてMACSを施した。
前記サイトカイン配合物は、CD4 T細胞を活性化する有効量内において、これを細胞培養液に添加する方法、時期、添加回数に特に制限されない。分離したCD4 T細胞を培養と同時に添加することもでき、細胞培養の後、一定の時間的な間隔をおいて添加することもできる。また、有効量内において単一投与することもでき、何回も多重添加することもできる。なお、サイトカインの各々が添加される時期も調節できる。望ましくは、あらゆるサイトカインを細胞培養と同時に単一処理するものである。サイトカインを添加した後、培地で1日から4日培養することが望ましい。
マウス(BALB/c、SLC Japan)の脾臓を取り出して、セル・グラインダー(cell grinder)で破砕した。破砕された細胞を、RPMI培地状態で1500rpmに遠心分離した後、RBC lysing buffer(Sigma、USA)10mlを加えて、常温で10分間反応させた。1500rpmに遠心分離した後、RPMI培地10mlで3回交換して、赤血球を除去した。得られた単核免疫細胞から、抗-CD4 T細胞抗体を使用して、MACS方法を用いて、CD4 T細胞を分離した。分離されたCD4 T細胞を、サイトカイン及び10%FBSを含むRPMI培地に入れて、48時間培養した。
1×104のサルモネラ菌(Salmonella typhimurium、高麗大学校)をマウスに経口投与して、バクテリア感染を誘発した。実施例1の方法にて活性化したCD4 T細胞を、PBS溶液に1×106になるように懸濁させ、サルモネラ菌に感染されたマウスに静脈注射した。
マウスの生存率を、PBSだけを投与した対照郡マウス、細胞株マクロファージ、または、CD4 T細胞を投与した実験郡マウスと比較した(表1)。
サルモネラ菌の感染後、実施例1の方法にて活性化されたCD4 T細胞を処理して生存したマウス郡(9匹)に対して、1×104のサルモネラ菌を経口投与して、感染を誘導した後、サルモネラ菌が感染されたことがなく、また、PBSだけが投与されたマウスに、上記と同様に、サルモネラ菌を経口投与した対照郡(9匹)との生存率の比較によって、CD4 T細胞の投与によるワクチン効果を見てみた。
細胞治療剤よるワクチン効果を測定するために、細胞治療の後、生存したマウスの血液を分離した。眼球から抽出したマウスの血液は、3000rpmに遠心分離して、血清を収得した。収得された血清に対して、抗原であるサルモネラ菌株に対する特異的な抗体反応の程度を、ELISA技法を使用して確認した。
本発明の方法にて活性化されたCD4 T細胞は、サルモネラ菌などのバクテリア感染疾患を効果的に予防又は治療することができる。
Claims (7)
- 個体の生物学的試料からCD4 T細胞を分離するステップと、
前記CD4 T細胞を、GM-CSF、IFN-gamma、TNF-alpha、Lectin及びIL-4を含むサイトカインを添加した培地で培養するステップと、を含む試験管内においてCD4 T細胞を活性化する方法。 - 生物学的試料が、血液(blood)、 血漿(plasma)、リンパ節(lymph node)、脾臓(spleen)、胸腺(thymus)、または、骨髄(bone marrow)である請求項1に記載の方法。
- サイトカインの濃度が、培地1ml当たり、0.05から0.2μgのGM-CSF、0.5から2μgのIFN-gamma、0.05から0.2μgのTNF-alpha、40から60μgのLectin、及び、0.05から0.2μgのIL-4である請求項1に記載の方法。
- 前記CD4 T細胞を、1日から4日培養する請求項1に記載の方法。
- 請求項1に記載の方法にて活性化されたCD4 T細胞を含むバクテリア感染疾患を予防又は治療する組成物。
- バクテリアが、サルモネラ(Salmonella)である請求項5に記載の組成物。
- バクテリアが、ネズミチフス菌(Salmonella typhimurium)である請求項6に記載の組成物。
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KR1020050033189A KR100735081B1 (ko) | 2005-04-21 | 2005-04-21 | Cd4 t 세포 활성화 방법 |
PCT/KR2006/000229 WO2006112588A1 (en) | 2005-04-21 | 2006-01-20 | Method for activating cd4 t cells |
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US (1) | US20080193422A1 (ja) |
EP (1) | EP1871871A4 (ja) |
JP (1) | JP2008538499A (ja) |
KR (1) | KR100735081B1 (ja) |
WO (1) | WO2006112588A1 (ja) |
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KR20150008306A (ko) * | 2013-07-12 | 2015-01-22 | 대한민국(관리부서 : 농림축산식품부 농림축산검역본부) | 인터페론-감마를 유효성분으로 포함하는 배지 조성물 및 이를 이용한 이식 거부 반응이 제거된 유도 만능 줄기세포의 제조 방법 |
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JP2002515756A (ja) * | 1997-01-31 | 2002-05-28 | ヘモソル インク. | 選択されたリンパ球の生産方法 |
JP2005503156A (ja) * | 2001-09-20 | 2005-02-03 | エクサイト セラピーズ, インコーポレイテッド | 細胞の活性化および増大 |
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US5374549A (en) * | 1991-01-31 | 1994-12-20 | Terumo Corporation | Process of enriching adherent CD4+ T cells from monocyte depleted peripheral blood mononuclear cells with interleukin 2 and interleukin 4 |
US5405751A (en) * | 1994-01-12 | 1995-04-11 | Schering Corporation | Prenatal diagnosis by cytokine-induced proliferation of fetal T-cells |
WO1999026658A1 (en) | 1997-11-24 | 1999-06-03 | Wong Johnson T | Methods for treatment of hiv or other infections using a t cell or viral activator and anti-retroviral combination therapy |
CA2341742A1 (en) | 1998-08-24 | 2000-03-02 | Maxim Pharmaceuticals, Inc. | Activation and protection of t-cells (cd4+ and cd8+) using an h2 receptor agonist and other t-cell activating agents |
AU2003202908A1 (en) | 2002-01-03 | 2003-07-24 | The Trustees Of The University Of Pennsylvania | Activation and expansion of t-cells using an engineered multivalent signaling platform |
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- 2006-01-20 US US11/912,148 patent/US20080193422A1/en not_active Abandoned
- 2006-01-20 EP EP06702936A patent/EP1871871A4/en not_active Withdrawn
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JP2002515756A (ja) * | 1997-01-31 | 2002-05-28 | ヘモソル インク. | 選択されたリンパ球の生産方法 |
JP2005503156A (ja) * | 2001-09-20 | 2005-02-03 | エクサイト セラピーズ, インコーポレイテッド | 細胞の活性化および増大 |
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EP1871871A1 (en) | 2008-01-02 |
WO2006112588A1 (en) | 2006-10-26 |
EP1871871A4 (en) | 2008-11-26 |
KR100735081B1 (ko) | 2007-07-06 |
KR20060110696A (ko) | 2006-10-25 |
US20080193422A1 (en) | 2008-08-14 |
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