JP6155271B2 - 複合サンプル中のil17陽性t細胞を同定するエピジェネティックマーカー - Google Patents
複合サンプル中のil17陽性t細胞を同定するエピジェネティックマーカー Download PDFInfo
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Description
1.多くの場合、測定される材料/サンプルは末梢血に由来するものではないことから、溶解性及び単一細胞懸濁液特性は満たされない。これは例えば、病理学的日常業務において行われるような全ての生検分析にも当てはまる。
2.これらの細胞を、その構造的完全性(「無損傷」)を維持するように、凍結するか、又はEDTA血として顆粒球等の亜画分が分解し始めるまで6時間を超えて保管してはならないため、検体が末梢血であっても無傷細胞を有するという必須条件は満たすことが困難である。
3.一般的な認識とは対照的に、全ての免疫細胞型に対する特異性の高い(表面)抗原は存在せず、したがって細胞型の同定は期待され得るほど明確ではない。
3a.抗原発現はデジタルプロセスではないため、細胞が陽性画分又は陰性画分のいずれに属するかを決定するのに閾値を規定する必要がある。T細胞では、この問題は特に顕著である:
配列番号2及び配列番号3は、図2による標的領域のヌクレオチド配列を示す。
配列番号4〜配列番号16は、実施例で使用したプライマー及びプローブのヌクレオチド配列を示す。
配列番号17は、ヒトIL17(A)のmRNAを開示する。
配列番号18〜配列番号22は、実施例3で使用される特に好ましいプライマー及びプローブのヌクレオチド配列を示す。
本発明者らは、T細胞を含む様々な血液サブセットを精製している。精製細胞に由来するDNAをバイサルファイト処理し、様々なCpGジヌクレオチドモチーフで分析した。次いで、本発明者らは、メチル化状態(元の配列で非メチル化されたシトシンについてのTに対する元の(ゲノム)配列でメチル化されたシトシンについてのC)を比較した。
特異的qPCRアッセイの開発
実施例1で得られた結果より、分析対象である好ましいCpG位置を含む目的のゲノム領域を同定した(アンプリコン1909、図2を参照)。
リバースプライマー:qPCR14 nmR2.1:GAGATGGATAAAATGTAGTGTTATT(配列番号5);
加水分解プローブ:qPCR14 nmP2.3:ACCCACTACAACACACCACATAAAT(配列番号6)。
qPCR14 nmF2.1:TTCTTCTATAACCTCATTAAAAACA(配列番号7)及び、
qPCR14 nmR2.2:ATGGATAAAATGTAGTGTTATTGT(配列番号8)。
qPCR14 nmF2.3:AACCCACTACAACACACCACA(配列番号9);
qPCR14 nmF2.4:ACCCACTACAACACACCACATA(配列番号10);
qPCR14 nmR2.3:AATGAGGTTTTTTTAGGAGTTATT(配列番号11);
qPCR14 nmR2.4:TGAGGTTTTTTTAGGAGTTATTG(配列番号12);
qPCR14 nmR2.5.TGGTTTAAATTAGTAAGAGTATTGTAT(配列番号13);
qPCR14 nmR2.6:GTTTAAATTAGTAAGAGTATTGTATGT(配列番号14);
qPCR14 nmP2.5:AAAAAACAATAACACTACATTTTATCCATCTCA(配列番号15)及び、
qPCR14 nmP2.6:TGAGATGGATAAAATGTAGTGTTATTGTTTTTT(配列番号16)。
最適化された特異的IL17A−qPCRアッセイの開発
このアッセイに対する特に好ましい「完璧な」プライマーシステムを開発するため、本来のバイサルファイト処理された配列に100%対応するものではないが、驚くほど特異性を高めた特異的なミスマッチを含むプライマーを開発した。
%IL17陽性T細胞=脱メチル化IL17Aコピー/全コピー×100
%IL17陽性T細胞=16.7/7996.67×100=0.21%。
qPCRアッセイ−オリゴヌクレオチド(5’→3’)
1.増幅プライマー
フォワードプライマーqPCR14 nmF2.1_M1:ATTCTTCTATAACCTCATTAAAAGCA(配列番号18);
フォワードプライマーqPCR14 nmF2.2_M1:TTCTTCTATAACCTCATTAAAAGCAA(配列番号19);
リバースプライマーqPCR14 nmR2.1:GAGATGGATAAAATGTAGTGTTATT(配列番号20);
リバースプライマーqPCR14 nmR2.2b:GATGGATAAAATGTAGTGTTATTG(配列番号21)。
PM−2.47nm:qPCR14 nmF2.1_M1+qPCR14 nmR2.1
PM−2.48nm:qPCR14 nmF2.1_M1+qPCR14 nmR2.2b
PM−2.53nm*:qPCR14 nmF2.2_M1+qPCR14 nmR2.1
PM−2.54nm:qPCR14 nmF2.2_M1+qPCR14 nmR2.2b
*は、この試験において最も良好な増幅効率を示す(特に好ましい実施の形態)。
qPCR14 nmP5:CCACTACAACACACCACATAAAT(配列番号22)
変更された反応条件(上記参照)として、好ましくは最大で6.4mMのMgCl2、すなわち5mMを超えるMgCl2を上記アッセイに使用することができた。
フローサイトメトリー及びバイサルファイト変換アッセイにより測定されたIL17A陽性細胞の割合
本発明者らは、PMA及びイオノマイシンによる刺激及び非刺激末梢血サンプルにおいてバイサルファイト変換を分析し、IL17A遺伝子の脱メチル化をモニタリングした。結果を、IL17A陽性細胞を検出するフローサイトメトリー分析と比較した(表2)。
Claims (14)
- 哺乳動物から得られた血液及び/又は組織サンプル中のIL−17陽性CD4陽性T細胞を同定する方法であって、
a)IL−17A遺伝子中の少なくとも1つのCpG位置のメチル化状態を分析することであって、
前記分析が、プライマー対の1つめのプライマーとして配列番号18又は配列番号19に示される配列からなるプライマーが選択され、プライマー対の2つめのプライマーとして配列番号20又は配列番号21に示される配列からなるプライマーが選択される、プライマー対での増幅を含み、
非IL−17血液細胞のCpG位置と比較したときの前記サンプル中の前記少なくとも1つのCpG位置の脱メチル化が、IL−17陽性CD4陽性T細胞の指標となること、及び
b)工程a)の分析に基づきIL−17陽性CD4陽性T細胞の量を定量化すること、
を含む、方法。 - プライマー対が配列番号19に示されるプライマー及び配列番号20に示されるプライマーの組み合わせである、請求項1に記載の方法。
- 前記メチル化状態の分析が、メチル化特異的酵素消化、バイサルファイトシークエンシングから選択される方法を含む、請求項1又は2に記載の方法。
- マーカーCD4、CD3、FOXP3及び/又はGAPDHの分析を更に含む、請求項1〜3のいずれか一項に記載の方法。
- 細胞の精製及び/又は濃縮の工程なしに行われる、請求項1〜4のいずれか一項に記載の方法。
- 全血及び/又はトリプシン処理されていない組織において行われる、請求項1〜5のいずれか一項に記載の方法。
- 前記サンプルが患者に移植されるサンプルである、請求項1〜6のいずれか一項に記載の方法。
- 哺乳動物におけるIL−17発現T細胞のレベルをモニタリングする方法であって、請求項1〜7のいずれか一項に記載の方法と、同定されたIL−17陽性T細胞の量を、同じ哺乳動物から採取された以前のサンプル及び/又は対照サンプルと比較することとを含む、方法。
- 前記哺乳動物が、乾癬性疾患、乾癬、乾癬性関節炎、関節リウマチのようなリウマチ性疾患、多発性硬化症、動脈硬化症、強直性脊椎炎、クローン病、炎症性腸疾患、炎症性疾患、ブドウ膜炎、肝炎疾患、狼瘡、肺疾患、喘息、高IgE症候群、抗腫瘍免疫、腎損傷、ウイルス性若しくは細菌性若しくは真菌性若しくは寄生虫性の感染症、内毒素性ショック、及び自己免疫疾患、ウイルス性感染症若しくは細菌性感染症、移植片拒絶反応、固形がん及び非固形がんを含むがん、及び/又はアレルギー若しくはIL−17発現T細胞に直接的に関連する任意の疾患等のIL−17媒介疾患及び抗IL−17療法の副作用を患っているか、又はその可能性がある、請求項1〜8のいずれか一項に記載の方法。
- 前記哺乳動物に与えた化学物質及び/又は生体物質に応じた前記IL−17発現T細胞の量を測定及び/又はモニタリングすることを更に含む、請求項1〜9のいずれか一項に記載の方法。
- 5mMを超えるMgCl2、又は最大6.4mMのMgCl2が増幅反応に使用される、請求項1〜10のいずれか一項に記載の方法。
- IL−17A遺伝子中のCpG位置のメチル化状態の分析に基づいて哺乳動物においてIL−17発現T細胞を同定、定量化、及び/又はモニタリングするためのキットであって、該キットは請求項1〜10のいずれか一項に記載の方法を実施するための材料を含み、
該材料は、
a)バイサルファイト試薬、及び
b)プライマー対の1つめのプライマーとして配列番号18又は配列番号19に示される配列からなるプライマーが選択され、プライマー対の2つめのプライマーとして配列番号20又は配列番号21に示される配列からなるプライマーが選択される、メチル化分析用プライマー対
を含む、キット。 - 哺乳動物におけるIL17発現T細胞の同定、定量化、及び/又はモニタリングのための請求項12に記載のキットの使用。
- プライマー対の1つめのプライマーとして配列番号18又は配列番号19に示される配列からなるプライマーが選択され、プライマー対の2つめのプライマーとして配列番号20又は配列番号21に示される配列からなるプライマーが選択される、プライマー対。
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PCT/EP2012/070676 WO2013057202A1 (en) | 2011-10-18 | 2012-10-18 | Epigenetic marker for the identification of il17 positive t cells in complex samples |
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