JP4890269B2 - Blimp1およびレポーター分子を共発現する改変細胞およびその使用方法 - Google Patents
Blimp1およびレポーター分子を共発現する改変細胞およびその使用方法 Download PDFInfo
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Description
発明の分野
本発明は、一般に、特定の系列の造血細胞およびそれらの分化段階を同定するモデルシステムに関する。さらに詳細には、本発明は、一般に、同定可能なシグナルを惹起することができるレポーター分子を共産生する(co-produce)ように改変された最終分化の遺伝子マーカーを有する、遺伝子改変細胞およびそのような細胞を含む非ヒト動物、ならびに、限定されるわけではないが、発達中の胚細胞、異常分化を伴う細胞(例えば、癌細胞)および造血細胞系列の細胞(例えば、Bおよび/またはT細胞など)などの細胞の分化またはトランスフォーメーション状態を調節することができる分子を同定する際のそれらの使用を提供する。同定された分子は、治療および予防用の薬学的組成物の基礎となる。
本明細書において参照する文献の詳細は、本明細書の末尾にも記載する。
本明細書を通して、その文脈が別様に求めていない限り、語「含む(comprise)」、または「含む(comprises)」もしくは「含んでいる(comprising)」などの語尾変化したものは、示されている要素もしくは整数または要素もしくは整数の群の包含を意味するが、任意の他の要素もしくは整数または要素もしくは整数の群の排除は意味しないことは理解されるだろう。
本発明は、造血細胞系の細胞もしくは胚性細胞を同定および単離するための、および/または造血細胞もしくは胚性細胞の分化をモニターするための方法の開発に、一部基づいており、この方法は、(レポーターによる)ポリペプチドの存在の検出または定量を含み、その存在は、細胞の最終分化に関連している。
* 488nm励起レーザーを使用
† バンドパス(bandpass)フィルタの幅
# ロングパス(longpass)フィルタ
(i)遺伝子改変細胞またはそのような細胞を含む非ヒト動物に薬剤またはワクチンを投与する段階であって、細胞または生物は、発現されるとき、レポーター分子と共発現される、Blimpまたはその一部、フラグメントもしくは機能的な形態を産生する、Blimpポリペプチドをコードする改変Blimp-1遺伝子を含む、段階;
(ii)その存在が、細胞表現型およびASCによる抗体産生を調節する該薬剤またはワクチンの能力の指標となる、レポーター分子の存在について細胞または生物を検査する段階。
(i)遺伝子改変細胞またはそのような細胞を含む非ヒト動物にワクチンを投与する段階であって、細胞または生物は、発現されるとき、レポーター分子と共発現される、Blimpまたはその一部、フラグメントもしくは機能的な形態を産生する、Blimpポリペプチドをコードする改変Blimp-1遺伝子を含む、段階;ならびに
(ii)その存在が、T細胞および/またはB細胞の活性化を調節するワクチンの能力の指標となる、レポーター分子の存在について細胞または生物を検査する段階。
Blimp-1突然変異対立遺伝子(Blimp gfp )の作製
イントロン3’からエキソン6に、スプライス受容体、3つ全てのリーディングフレーム内の停止コドン、内因性リボソーム認識部位(IRES)、eGFPをコードするcDNA、および転写を停止させるSV40ポリアデニル化シグナルからなるeGFP発現カセットが挿入されている、Blimp-1ターゲティング構築物を産生させた。組み込まれたターゲティングベクターでの胚性幹(ES)細胞の選択を可能にするために、PGK-Neo γ 遺伝子もそのイントロンに挿入されている。それらのeGFPカセットおよびNeo γ カセットをFrt部位に隣接させて、挿入されたDNAのflpリコンビナーゼ媒介欠失を可能にする。Blimp-1ターゲティング構築物、G418により選択される耐性クローンを用いてC57BL/6 ES細胞をエレクトロポレーションし、5’および3’ゲノムDNAプローブへのサザンハイブリダイゼーションによりスクリーニングした(図1C)。Blimp gfp 対立遺伝子を有する4つの正確にターゲティングされたクローン(図1C)を、300個のスクリーニングされたクローンから同定した。これらをBALB/c胚盤胞に注入して、キメラ創始者を得た。これらのキメラを増殖させ、生殖系伝達を1つのクローン(4F3)で達成した。
Blimp-1の発現パターンの記述を可能にするGFPレポーター
当初、Blimp-1は、ASC分化を受けるように誘導されたBリンパ球においてのみ発現されると報告された(Turner et al., (前記))。しかし、その後の研究により、胚形成中(Chang et al., Mech Dev 117:305, 2002)、および骨髄細胞における(Chang et al., (前記), 2000)Blimp-1のより幅広い発現パターンが明らかになった。Blimp gfp 対立遺伝子により、造血系列の中でも、より広くは生物においても、Blimp-1の発現パターンをより完全に定義することができる。上で概説したターゲティング戦略は、結果としてBlimp gfp 対立遺伝子を生じさせ、それが、内因性Blimp-1調節要素の制御下で2シストロンのmRNAからGFPを発現し、従って、完全Blimp-1発現パターンを反復すると予測される。加えて、この戦略は、Blimp-1 mRNA転写産物を分断して、DNA結合モチーフを含有するジンクフィンガードメインを欠くトランケートされたバージョンのBlimp-1タンパク質(エキソン1〜6)を産生させる。これに従って、インビトロでLPSを用いて分化するように誘導されたBlimp gfp/+ B細胞のウエスタンブロッティングは、野生型Blimp-1タンパク質バンドおよびトランケート型Blimp-1タンパク質バンドの両方を示した(図1D)。生細胞におけるGFP発現および固定された組織におけるBlimp-1タンパク質のモニタリングにより、インビボおよびインビトロでのBリンパ球の遺伝子活性および分化の運命を単細胞レベルでモニターすることができる。
ASCにおけるBlimp-1のインビボ発現
Blimp gfp/+ マウスにおけるリンパ組織の検査は、高Blimp-1の小集団が、骨髄(0.1〜0.2%)、脾臓(0.4〜0.6%)およびリンパ節(0.1%)において細胞を発現していることを示した(図2)。さらに、GFP+細胞の表現型解析は、それらが、以前に定義された高Synd-1/低B220のASC集団、ならびに以前に十分に特性付けされていなかった低Synd-1〜Synd-1陰性の表現型を表すことを示した(図2、(Underhill et al., (前記))。これらの細胞がASCであることを確認するために、Blimp gfp/+ 骨髄および脾臓からGFP+細胞を選別し、Ig産生についてのELISpot分析に供した。図3からわかるように、細胞の75〜100%がIg分泌性細胞であった(3回の独立した実験の代表)。さらに、GFP陰性画分の選別は、0.001%のASCの頻度(細胞100,000個あたり1個未満)、これに対して、未選別骨髄におけるこれらの細胞の頻度は、0.05〜0.09%であった(100,000個あたり50〜90個)。従って、Blimp gfp 発現性ASCの単離は、未分別細胞の100,000倍の濃縮をもたらし、これらの希少細胞を単離するための実質的に決定的な方法をもたらす。加えて、GFP+ ASC集団における全てのIgアイソタイプを表した(図3)。
インビトロで誘導されるASCにおけるBlimp-1の発現
ASC系列への決定付けおよびASC系列までの進行に影響を及ぼすパラメータをインビトロで定量分析する方法論を開発した。このシステムは、パーコール(Percoll)勾配遠心分離および磁性ビーズ濃縮により精製し、B細胞増殖およびASCへの分化を誘導する様々な刺激の存在下で培養する、小さな休止B細胞の単離を含む。これらの条件としては、IL-4および抗CD40を使用するT依存性応答の模倣またはLPSを使用するT非依存性反応の模倣が挙げられる。加えて、IL-5を濃度調節しながらこれらの培養物に滴下して分化速度を加速し、および抗IgD(1.19)架橋を行って、抗原特異的応答を活性化することができる。1〜5日目にフローサイトメトリーにより培養物を分析して、Blimp gfp およびSynd-1 + 発現性細胞の頻度を測定した。培養物中のASCの数は、ELIspotにより判定した。
Blimp-1は胚形成に必要とされる
ホモ接合のBlimp gfp/gfp 動物を産生するために、Blimp gfp/+ 個体を交雑させた。Blimp-1野生型およびBlimp gfp 特異的PCRプライマーを使用して、これらの交雑からの子孫を生後21日目に遺伝子型解析した。Blimp gfp/+ マウスが、生存しており健康であったのに対し、Blimp gfp/gfp 個体は、同定されなかった。これは、Blimp-1欠失が、結果として胚性致死または早期産後致死を生じさせることを示している(図7)。Blimp gfp/gfp 動物が死亡する段階により近い段階を検査するために、Blimp gfp/+ マウスの時限交雑から生じた胚を検査した。これらのデータは、Blimp gfp/gfp 胚が、胚性段階E15.5ほどもの後期に生存していることを示す。しかし、生存能力がある、より高い齢の個体が、文献に記載されたことはない。Blimp-1が胚形成中に広範に発現されることは公知であり、この発見は、Blimpgfpマウスを使用する分析により支持されている。
Blimp-1は、抗体産生に不可欠である
Blimp gfp/gfp 動物の胚性致死を回避するために、および抗体産生におけるBlimp-1の重要性を直接検査するために、致死量の放射線を照射した同系マウスの胎仔肝臓幹細胞再構成を用いて、造血形全体にわたって機能的なBlimp-1タンパク質を欠く成体マウスを産生させた。これらのBlimp gfp/gfp キメラ動物は、健康であり、検査した全ての造血細胞系列を比較的正常な数、含有している。LPSまたはCD40L/IL-4およびIL-5、いずれかでの刺激後のこれらのマウスにおけるASC集団のインビトロ分析は、主としてsynd-1+であるGFP+ Blimp欠失細胞の存在を示した(図8A)。重要なことに、ELIspotアッセイにより評価したところ、これらの細胞は、抗体を分泌できなかった(図8B)。従って、Blimp gfp 、ここに記載するマウスモデルは、ASCを単離するための決定的ツールをもたらすばかりでなく、ホモ接合突然変異Blimp gfp/gfp 脾細胞からのBlimp-1発現性細胞の集団の同定を可能にし、それにより、Blimp-1欠失の表現型の根底にあるメカニズムの分析を大いに助長する。
他の造血系列におけるBlimp-1の発現
Blimp gfp レポーター系により、初めて、造血におけるBlimp-1の発現パターンも定義することができた。上で述べたように、Blimp gfp マウスのリンパ器官の分析は、GFP高産生性集団が、ほぼ排他的にASCであることを示した。しかし、低レベルのGFP産生性細胞も出現した。
Blimp gfp マウスを使用する、Blimp-1の癌における役割の検査
ASC分化の検査におけるその有用性に加えて、Blimp gfp レポーターマウスは、この細胞タイプの悪性トランスフォーメーションを検査するために使用することができる。マウスでは形質細胞種およびヒトでは多発性骨髄腫と呼ばれるASCの腫瘍は、IgHイントロン性エンハンサーの制御下、B細胞系列においてv-abl癌遺伝子を発現するEμ-v-ablトランスジェニックマウス(Rosenbaum et al., (前記))において、特異的におよび頻繁に惹起される。これらのマウスをBlimp gfp 突然変異マウスと交雑させて、腫瘍の潜在性および発病率に対するBlimp-1遺伝子の一方または両方のコピーの欠失の影響を判定した。2つの結果が考えられる:Blimp-1は、形質細胞分化プログラムを誘導することにより、v-ablトランスフォーメーションの機会に関するウインドウを開くために必要となり得る。このトランスジーンは、初期B細胞における発現にもかかわらず、形質細胞種しか誘導しない(Rosenbaum et al., (前記))。従って、機能的Blimp-1対立遺伝子の欠失により、腫瘍発病率の低下または潜在性の増加が予測されよう。または、大きな比率のv-abl誘導形質細胞種が、再配列されたおよび活性化したc-myc遺伝子も有するので、Mycは、トランスフォーメーションにおける本質的な共同作用性活性であり得る(Rosenbaum et al., (前記))。Blimp-1は、最終ASC分化の間のc-myc発現を抑制すると一般に考えられている(Lin et al., Science 276:596, 1997)。このシナリオでは、機能的Blimp-1の欠失が、持続性c-myc発現を可能にすることとなり、それにより、形質細胞種の発現が加速し得る。
T細胞における最終分化の調節におけるBlimpの役割を評価する方法
マウス
Blimp gfp マウス(Kallies et al., J Exp Med 200:967-977, 2004)、Rag1 -/- マウスおよびRag2 -/- マウスを、C57BL/6バックグラウンドで保持した。Blimp gfp 遺伝子型解析および胎仔肝臓キメラを、記載されている(Kallies et al., 2004 (前記))とおり作製した。
CD4(GK1.5)、CD8(53.6.7)、TCRβ(H57-597)、Ly5.2(ALI-4A2)に対するモノクローナル抗体(mAb)を、プロテインG-セファロースカラム(Amersham Pharmacia Biotech)でハイブリドーマ上清から精製し、ビオチン(Pierce Chemical Company)、アロフィコシアニン(APC)およびフィコエリトリン(PE)(ProZyme)に、推奨どおりにコンジュゲートさせた。ビオチン化抗CD25(7D4)およびCD122(Tm-β1)ならびにPEコンジュゲート型抗CD44(IM7)、CD62L(MEL-14)、IFNγ.□□□〜 1□.、IL-4(11B11)は、PharMingenから入手した。ビオチン化mAbは、ストレプトアビジン-PEまたはCy5と共に発現された(Southern Biotechnologies Inc.)。LSRサイトメーター(BD Bioscience)で細胞を分析し、高速フローサイトメーター(Moflo cytomation and BD Biosciences)で細胞選別を行った。サイトカインについての細胞内染色は、当技術分野において公知の標準的な手順に従って行った。IFNγおよびIL-10産生についてのELISAは、記載されている(Brady et al., J Immunol 172: 2048-2058, 2004)とおり行った。IL-4 ELISAは、1つのモノクローナル抗体を捕捉試薬として使用し、第二のモノクローナル抗体を検出に使用した。ELISAは、三回重複で行い、組換えタンパク質標準物質を使用して定量した。
例えば、Rosenbauerら(Embo J 21:211-20, 2002)が記載したように、全タンパク質抽出物を相当数の細胞から産生させ、ウエスタンブロッティングを行った。抗Blimp-1 mAbは、以前に記載されている(Kallies et al., 2004 (前記))。抗Vav1を使用して、等量のタンパク質負荷を確認した。
20μLのPBSで希釈した4×105単純疱疹ウイルス(HSV-1 KOS株)にマウスを感染させた。後ろ足の足蹠と踵の間への皮下注射によって投与した。感染したマウスの脾臓および膝窩リンパ節を、その後、分析のために回収した。
gB特異的細胞毒性T細胞リンパ球(CTL)を常用の手順[Belz, 2001]により作製した。感染したマウスから脾臓を除去し、108個の1000Gy照射gB498-505被覆C57BL/6脾細胞を5日間単細胞培養した。細胞毒性は、従来の51Cr放出アッセイで評価した。EL4(H-2b)標的細胞を、Na51Crで1時間標識し、gBペプチドを60分間適用し、2回洗浄し、5,000標的/ウエルでプレーティングした。その後、それらをエフェクター集団と共に5時間インキュベートし、その後、γ計数用に上清を回収した。二倍リンパ球希釈物を三回重複でアッセイし、一方、未処理およびTriton X-100破壊対照は、四回重複で測定した。特異的溶解率は、100×(エフェクターを伴う標的からの51Cr放出-標的のみからの51Cr放出)/(Triton X-100を伴う標的からの51Cr放出)として計算した。T細胞不在下でインキュベートした標的からの51Cr放出レベルは、全Triton X-100媒介51Cr放出の10%未満であった。
H-2Kb糖タンパク質と単純疱疹ウイルスの糖タンパクB(gB498-505)由来のペプチド(SSIEFARL)とのMHCクラスI四量体型複合体[Altman, 1996 #102; Allan, 2003 #100]を使用して、ウイルス特異的CD8+ T細胞を同定した。カルボキシ末端膜貫通ドメインがbirAビオチン化モチーフで置換されている組換えH-2Kb分子を、ヒトβ2-ミクログロブリンおよびウイルスペプチドでリフォールディングし、birAでビオチン化し、4:1のモル比でニュートラビジン(neutravidin)-PE(オレゴン州、Eugene、Molecular Probes)と複合させた。室温で、60分間、リンパ球をPBS/BSA/アジド中の四量体型複合体について染色し、その後、抗CD8αAPCで染色し、2回洗浄し、フローサイトメトリーによって分析した。
器官を10%緩衝ホルマリン中で固定し、パラフィンに包埋し、薄片に切り、ヘマトキシリン/エオシンで染色した。
Blimp発現は、IL-21により誘導することができ、ナイーブCD4+およびCD8+ T細胞の活性化エフェクター細胞への成熟に関する重要な成分であり、正常なリンパ球恒常性に不可欠である
Blimpは、全てのリンパ球における保存的な最終分化プログラムの主調節因子である
要約すると、本明細書において記載のデータは、Blimp-1が、活性化された従来のT細胞において様々な状況下で発現されることを実証している。Blimp-1欠失T細胞を注射した、または突然変異幹細胞で再構成されたマウスが、進行性多器官リンパ球増殖性疾患の結果として死亡するので、Blimp発現は、正常なリンパ球恒常性には不可欠である。加えて、T細胞分化の恒常性を調節することが公知であるIL-21などのサイトカインは、Blimp-1発現の強力な誘導因子であり、Blimp-1不在下、インビトロで強化増殖を支持した。従って、Blimp-1発現は、初期刺激によってではなく、免疫応答の完了に向けて、エフェクターT細胞の分化を誘導し得る。従って、Blimp-1は、T細胞収縮の遺伝プログラムおよび/または免疫恒常性に不可欠である記憶形成を調節するように機能する、同定された最初の転写因子である。
Claims (26)
- 内因性Blimp調節要素の制御下で発現され、かつ、レポーター分子と共発現されるBlimp(PRDM-1)を産生する、Blimpポリペプチドをコードする改変Blimp(PRDM-1)遺伝子を含む遺伝子改変細胞を含む非ヒト生物であって、細胞内におけるBlimpの存在が、細胞表現型および/または細胞における最終分化への決定付け(commitment)に関連し、かつ、該細胞が抗体分泌細胞(ASC)である、生物。
- 改変Blimp遺伝子が、Blimpコード配列およびレポーター分子コード配列をコードするBlimp mRNA転写産物をコードする、請求項1記載の生物。
- レポーター分子コード配列がBlimp対立遺伝子のイントロン内に挿入される、請求項2記載の生物。
- 改変Blimp対立遺伝子がホモ接合またはヘテロ接合の形態で存在する、請求項3記載の非ヒト生物。
- 改変Blimp対立遺伝子がヘテロ接合の形態で存在する、請求項4記載の非ヒト生物。
- 改変Blimp対立遺伝子が、機能的Blimp転写因子をコードする、請求項1〜5のいずれか一項記載の非ヒト生物。
- 非ヒト霊長類、家畜、愛玩生物、実験検査生物、爬虫類または両生類などの任意の生物に由来する細胞または遺伝物質を含む、請求項1記載の非ヒト生物。
- 齧歯動物(マウスを含む)、モルモット、ブタ、カモ、ウサギまたはヒツジなどの実験検査動物に由来する、請求項7記載の生物。
- レポーター分子の検出が細胞表現型および/または最終分化への細胞の決定付けの指標となる、請求項1〜8のいずれか一項記載の生物。
- 胚、生殖体またはES細胞の形態で提供される、請求項1〜9のいずれか一項記載の非ヒト生物。
- レポーター分子が蛍光または発光レポーター分子である、請求項1〜10のいずれか一項記載の生物。
- 内因性Blimp調節要素の制御下で発現され、かつ、Blimpおよびレポーター分子を共発現する、Blimpタンパク質をコードする改変Blimp遺伝子を含む遺伝子改変造血細胞を含む非ヒト動物をスクリーニングする段階を含み、レポーター活性の検出が、細胞表現型および/または最終分化への細胞の決定付けの指標となる、造血系の細胞を表現型解析および/またはモニタリングする方法であって、該細胞がB細胞系列の細胞の集団において同定または単離された抗体分泌細胞(ASC)である方法。
- 細胞の表現型解析および/またはモニタリングが蛍光または発光レポーター分子のサイトメトリー分析により達成される、請求項12記載の方法。
- レポーター活性を示さない細胞の中から、レポーター活性またはレポーター活性もしくはレベルの変化を示す細胞を単離または選択する段階をさらに含む、請求項12記載の方法。
- レポーター活性細胞の単離が、フローサイトメトリー、レーザー走査サイトメトリー、クロマトグラフィーおよび/または他の同等の手法によるものである、請求項14記載の方法。
- さらなる選択マーカーを使用してレポーター活性細胞を選択する段階をさらに含む、請求項14記載の方法。
- 以下の段階を含む、ワクチンの抗原性または免疫原性を検査する方法であって、下記段階における細胞が抗体分泌細胞(ASC)である方法:
(i)請求項1に記載の非ヒト生物にワクチンを投与する段階;
(ii)その存在が造血細胞において最終分化を誘導するワクチンの能力の指標となるレポーター分子について、該生物を検査する段階。 - レポーター活性の存在が抗体分泌細胞(ASC)において最終分化を促進するワクチンの能力の指標となる、請求項17記載の方法。
- 以下の段階を含む、造血細胞における最終分化の作動薬(agonist)または拮抗薬(antagonist)についてインビトロまたはインビボでスクリーニングする方法であって、該細胞が抗体分泌細胞(ASC)である方法:請求項1に記載の非ヒト生物に1つまたは複数の薬剤を暴露する段階;ならびに、その存在が、最終分化を作動し、または最終分化に拮抗する1つまたは複数の薬剤の能力の指標となるレポーター分子の存在またはレポーター分子のレベルの変化について、該生物を検査する段階。
- 細胞が、Blimpコード配列およびレポーター分子コード配列をコードする、Blimp mRNA転写産物をコードする改変Blimp遺伝子を含む、請求項12、17または19いずれか一項記載の方法。
- レポーター分子コード配列がBlimp対立遺伝子のイントロン内に挿入される、請求項20記載の方法。
- 改変Blimp対立遺伝子がホモ接合またはヘテロ接合の形態で存在する、請求項21記載の方法。
- 改変Blimp対立遺伝子がヘテロ接合の形態で存在する、請求項22記載の方法。
- 改変Blimp対立遺伝子が、機能的Blimp転写因子をコードする、請求項20記載の方法。
- 細胞が、非ヒト霊長類、家畜、愛玩生物、または実験検査生物、爬虫類または両生類などの任意の生物に由来する、請求項20記載の方法。
- 実験検査生物が、齧歯動物(マウスを含む)、モルモット、ブタ、カモ、ウサギおよびヒツジから選択される、請求項25記載の方法。
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