この研究は、ウィスコンシン州とウィスコンシン州の子どもの研究所の少年Diabetesat医科大学のマックスマギー国立研究センターによってサポートされていました。資金提供者は、研究デザイン、データ収集と分析の役割、または原稿の準備がなかった。

1。抑制アッセイを設定する前に、その後の細胞の刺激とするための抗ヒトCD3（クローンUCHT1、最終濃度1μg/mlの）でコーティングtosylactivatedビーズへの1つのニーズは、ビーズが効率的に人間を用いた in vitro増殖アッセイで設定することによって被覆されているかどうかを確認するT細胞<ol><li>オリジナルバイアル、磁気スタンドの場所からM - 450 tosylactivatedビーズを1mlを取り、すべてのビーズがチューブの側面に付着するまで押し続けます。チューブは、磁気スタンドに入れたままでバッファを削除します。 37、撹拌機を再懸濁しますビーズをbuffer1の1mlの抗ヒトCD3の40μlのを追加°、磁気スタンドからチューブを取り、buffer1の1mlを追加、再び磁気スタンドにチューブを配置し、チューブを磁気スタンドにある間buffer1を削除するC 15分間、0.1％w / vのBSAを追加し、次の16時間攪拌を続行。 </li><li>バッファを削除するとbuffer2の1mlのビーズを再懸濁さ;ビーズがある℃、一回5分間buffer3で2〜8℃上記で説明したように磁気スタンドを使用して2〜8℃で5分間二回buffer2でビーズを洗浄4 × 10 8 / mlの最終濃度、使用できるようになります。 </li><li>分注し50,000 25000 PBMC /ウェル、96ウェルプレートでトリプリケートでとCD3でコーティングされたビーズの数が可変（4 × 10 8ビーズ/ mlと、CD31μg/mlの、例えば1、2、3、4、5ビーズ/細胞追加）細胞当たりのビーズの最適数を決定するために。文化の中で72時間後、1μCi[3 H]チミジンを追加し、37℃でインキュベートし、次の16時間継続する。マルチスクリーン収穫プレート（ミリポア）で収穫細胞は、シンチレーション液を追加し、トップカウントNXT（パッカード、CT）を使用しても/分当たりのカウント（cpm）をお読みください。抑制機能を失う可能性が制御性T、の過剰刺激を避けるために、ビーズ/インプレッション単価が5000を超えている細胞の比率が15000未満を使用してください。通常、3ビーズ/細胞の比は、これまでに1から4までテストしたすべての被験者群でのレスポンダとTreg細胞の両方を刺激する。 </li></ol> 2。地元の輸血センターから健康なドナーからのまたは人間leukopacksまたはバフィーコート（BC）からの全血からPBMCを分離、通常健康なボランティアから採取し、無料で利用できる（図1） <ol><li> BC（〜50ml）にPBS 1:6を（250ミリリットルを追加する）希釈してください。今すぐ300ミリリットルの合計体積があります。徐々に15ミリリットルをFicoll - Paqueの上に希薄化後のBCの25ミリリットル層PLUSは、層を乱すことなく、50ミリリットルのファルコンチューブに加えた。 4℃で30分間800xg（スイングバケットローターSH - 3000とソーバル遠心機で1400rpm）で遠心° C、ブレーキをオフにした。 </li><li>慎重にPBMC層（中間相）を収集し、新鮮な50ミリリットルファルコンチューブに移す。 DPBSで50ミリリットルにチューブが満杯になってPBMCを洗ってください。つのチューブに2細胞ペレットを収集する。で10分間4℃で400xgで遠心分離、二回一本のチューブに2細胞ペレットを組み合わせたたびに洗浄ステップを繰り返します。シングル50ミリリットルチューブにすべての細胞ペレットを組み合わせる。 </li><li>トリパンブルー排除試験を用いてPBMCを数えます。染色トリパンブルーの180μlにPBMCの20μlのを追加してトリパンブルーで1:10希釈液を加えます。よく混和して、直ちに血球計でカウントする20μlのかかります。二つの四角いブロック16より小さい正方形を含むそれぞれの内にあるすべての未染色とのみ青色に染色された細胞数を数える。両方の数値の平均を取ると10を掛けます。 PBMC / mlの数を得るために100でこの数を分け。生存細胞の割合として計算する[1 - （全細胞の青色のセル/数の数）× 100]。生存率が≥95％の場合進みます。 </li></ol> 3。 CD4 T細胞のMACS事前ソート<ol><li>先に進む前に、新しいチューブにPBMCの1mlを転送する、5000rad - これらは、抗原提示細胞（APC）になると照射のために細胞を調製するためにメディアの4ミリリットル加える。 </li><li> 4℃で10分間PBS/2mM EDTA/0.5％BSAバッファー中で250xgでの細胞の残りを遠心分離℃にPBS/2mM EDTA/0.5％BSAバッファーの4ミリリットルで上清、再懸濁します細胞を捨て。 </li><li> MACS抗CD4マイクロビーズ200μlのを追加し、4℃でインキュベート℃で20分間。 </li><li> 4℃で10分間250xgでPBS/2mM EDTA/0.5％BSAバッファーと遠心の40ミリリットルを追加することで、洗う℃に脱気、室温PBS/2mM EDTA/0.5％BSAバッファーの8ミリリットルの上清と再懸を捨て。 </li><li> LSカラム上にロードする前に、プレセパレーションフィルターを使用してフィルタ - 別々の細胞懸濁液。 </li><li>キャリブレーションLSカラムに各細胞懸濁液および層の4ミリリットルを分割（deggassed PBS/2mM EDTA/0.5％BSAバッファーの3ミリリットルを持つ）。 LSカラムはMidiMACSセパレータに固定されています。細胞懸濁液は、再実行のフロースルー、不足またはdeggassed室温PBS/2mM EDTA/0.5％BSAバッファーの3ミリリットルを加えるとバッファがから実行できるようにする前に。それが出て明らかになるまでより多くのバッファを追加。 </li><li> LSのカラムにピペット5ミリリットルdeggassedバッファ、〜1.0ミリリットルを介して実行させる新しい滅菌15ミリリットルコレクションチューブにMidiMACSセパレータと場所からLSカラムを削除します。列にプランジャーを入れて、ゆっくりと残りの部分を押してくださいボリュームから。 </li><li>両方のLSの列でも同じことが二CD4 +分画を組み合わせる。 50ミリリットルPBSとカウントセルを追加します。予想される収量は、5 × 10 8細胞までです。 2ミリリットルPBS/2mM EDTA/0.5％BSAバッファー中でも10分、懸濁し、細胞のために400xgで遠心分離します。 </li></ol> 4。蛍光活性化細胞選別（FACS）の分離（図2） <ol><li> 、抗ヒトCD8 - FITC（クローンRPA - T8）、抗ヒトCD14 - FITC（クローンM5E2の20μlを、20μlの次の細胞表面マーカー（光から保護保持）にCDのマーカーに対する抗体のカクテルを作るLPS受容体）、抗ヒトCD32 - FITC（クローンFLI8.26の20μlを、FcγRのタイプII）と抗ヒトCD116 - FITC（クローンM5D126μlの、GM -CSFRαチェーン）とは、代わりに、40μlの抗を追加ヒトCD4 - APCCy7（クローンRPA - T4）。 </li><li> 5μlのは、2ミリリットルの細胞懸濁液を取り出し、汚れカクテルの2μlのと染色、これは、しきい値（蛍光色素マイナスOne - FMO）5を決定するためのチューブです。 </li><li>染色カクテルに、そして4℃で30分で細胞懸濁液とインキュベートするカクテルを追加し、抗ヒトCD25 - PE（IL -2RαクローンM - A251）の50μlを追加。 PBS緩衝液、10分および10 7 / mlの細胞濃度に懸濁し、細胞のための400xgで遠心分離で細胞を洗浄。 </li><li>未染色の細胞と細胞またはFACSアリア（BD Biosciences社、サンノゼ、ニュージャージー州）での細胞選別のために補償制御として使用する単一の蛍光色素で染色されたビーズを準備します。 </li><li>ユーザーは、CD25 + T細胞（図2a）のソートのしきい値を設定できるようになりますFMOチューブに細胞を取得する。単球、マクロファージおよび他のすべてのCD4 + -非T細胞（図2b）からなるFITC陽性細胞を除外するためにFITC -陰性細胞の周りのゲートを設定します。 </li><li>別のプロットでは、CD25 -、CD25lowとCD25high T細胞- Tregsの（CD25の最大数を発現する細胞の上位1％）（図2c）のためにゲートを描きます。細胞サブセットは一般的に高純度（図2d、2eと2fを）示しています。 </li><li> 10分間400xgで細胞を用いたコレクションチューブを遠心し、メッキまで氷上で保管して下さい。 </li></ol> 5。 200μl/wellで96穴プレート（表1として添付する方式）で細胞培養を設定する<ol><li> CD3でコーティングされたビーズのアリコート50μlの（1μg/ mlの）は3ビーズ/ U -底部では10％プールしたヒトAB血清で完全なメディアに再懸濁してウェル内のレスポンダのセル、96ウェルプレートになるように計算。 （たとえば、CD3でコーティングした2ミリリットルのメディアを作るために、CD3でコーティングされたの株式ビーズ-最終濃度4 × 10 8 / mlから7.5μl取るとメディアの2ミリリットルで希釈し、すべての50μlのは75,000 beads-3beads/cellが含まれます）。 </li><li> 5 × 10 5 / mlの細胞濃度と、&quot;唯一のTregsの&quot;というラベルの井戸を含む、以前に追加した刺激、との各ウェルに（2.5 × 10 4細胞を含む）50μlずつを追加し、&quot;APCだけ&quot;と、表の&quot;メディアのみ&quot;に照射APCを薄める1。 </li><li>表1の設計は、次の連で2.5 × 10 4 /ウェルCD4CD25 -またはCD4CD25low T細胞を追加。 </li><li>比1:10（2,500 Treg細胞）の共培養（列B、表1）にし、&quot;唯一のTregsの&quot;というラベルのウェルにTregsのを追加し、5％CO 2とCO 2インキュベーターで37℃プレート℃でインキュベートする、72時間飽和湿度インチ</li><li>パルス1μCi[3 H]チミジンをウェルの中とは° C次の16時間37℃でインキュベーションを続ける。 </li></ol> 6。収穫とカウント<ol><li>パッカードfiltermate収穫機または別のシステムを使用してマルチスクリーン収穫プレート上に細胞を回収。 </li><li>シンチレーション液（Microscint 20）追加、最後のステップの準備のために透明なプラスチックカバーでプレートを収穫するカバー。 </li><li>分当たりのカウント数（cpm）を読んで/ウェルトップカウントNXT（パッカード、CT）または別のシステムを使用して。 </li></ol> 7。抑制のコンピューティングの割合<ol><li>細胞は三連で培養したように、平均を各条件ごとに計算されます。変動係数が&gt; 30％の場合、外れ値が排除され、2つの井戸からのみCPMは平均化されます。抑制の割合は、共培養の単文化とc = CPMの計算[（SC）/秒] × 100％、S = CPMすることによって得られる。 </li><li>ナイーブ（CD25 -）とin vivoで活性化（CD25low） にエフェクターT細胞は、レスポンダーT細胞として播種され、これらのサブセットの各々を抑制する制御性Tの能力の差が前提に基づいて潜在的な機能的な予後の指標として取り込まれ、使用されているようにその活性化細胞では抑制が困難です。 </li></ol> 8。代表的な結果： 方法に大きなばらつきが使用され、in vitroでヒトの抑制アッセイから得られた結果は、私たちはアッセイ1に影響を与える条件の総合的な研究を実行するためのプロンプトが表示。我々は以前の6に決定Teffs（1:10）、：私達はTregsの間に低比を考慮するだけでなく、Tregの機能をテストする検定だけでなく、その純度を開発している。さらに、Tregsのは目に異なる免疫バランスが侵害された場合T1D開発するためにリスクのある被験者のように成功し、ナイーブと、図3に、より顕著になる私たちの以前の研究2,4に示すように、さらに健常者の in vivo活性化T細胞における抑制するEIRの能力。アッセイは、LS（最近発症（RO）T1Dと長年、私たちは健康な制御との間でそれらの機能を比較することが、我々は自然（nTregs）の抑制機能を比較して研究、誘導（iTregs） と in vitro拡張のnTregs で非常にうまくいった）T1D科目。我々はRO T1D被験者はLS T1Dと健常者7に比べて機能的iTregs両方と拡張nTregsを生成するのより良い能力を持っていたと結論づけた。したがって、このアッセイは免疫不均衡の初期および後期の状態の両方の認識に優れたツールとして使用することができます。 in vitroでの抑制アッセイのために必要な手順のスキーム1回路図のプレゼンテーション<img src="/files/ftp_upload/3071/3071fig1.jpg" alt="図1"/> 図1写真を提示in vitroでの抑制アッセイのステップ<img src="/files/ftp_upload/3071/3071fig2.jpg" alt="図2"/> FACS細胞の分離2。ゲーティング戦略図 。 ）CD25 +しきい値は、FITC -陰性細胞は（さらにゲートしたとCD + CD25 -、CD + CD25lowとCD + CD25highとして収集された）細胞は、C FITC陰性としてゲーティングした）蛍光色素マイナスワン（FMO）、Bに応じて調整された割合、dで示すように、Tregsのは））、電子、ソート後にTregsのをソートFACS）ソート後にソートされたCD + CD25lowをFACS、およびf）ソート後にソートされたCD4 + CD25 - T細胞をFACS <img src="/files/ftp_upload/3071/3071fig3.jpg" alt="図3"/> 図3。健常者の代表の結果）関連するすべての細胞サブセットのための単一文化として提示健常者（ナイーブ- CD25 -、in vivoで活性化- CD25low で 、抗原提示細胞- APCの毎分のカウントの代表的な結果（CPM）がと各CD25 -と自己制御性TによってCD25lowレスポンダーT細胞は健常者のために提示されている（nの抑制の制御性T細胞- Tregの）だけでなく、レスポンダーT細胞の共培養（CD25 -またはCD25low）とTregsの。B）の割合= 4）。抑制は、共培養の単文化とc = CPMで[（SC）/秒]として× 100％、S = CPMを計算した。レスポンダーT細胞を抑制する制御性Tの容量のわずかな違いが気づいていたが、それは。C（t検定P = 0.08ペア））発表は、応答側としてCD25 -とCD25lowを含め、それぞれ単一の文化のためのリスクの被験者でのcpmである有意ではなかったT細胞だけでなく、それぞれのレスポンダーT細胞サブセットは、自家で各CD25 -とCD25lowレスポンダーT細胞の抑制の制御性T（CD25-/TregsとCD25low/Tregs）。d）の割合で播種されたAPCとTregsの、そして共培養Tregsのは、リスクの被験者（N = 4）でのために提示されます。 CD25 -対CD25lowレスポンダーT細胞を抑制する制御性Tの能力の差は有意であった（t検定P = 0.04をペアになった）。 表1。回路図は 、in vitro 抑制アッセイのセットアップ<table border="1"><tbody><tr><td></td><td> 1月3日</td><td> 4月6日</td><td> 7月9日</td><td> 10月12日</td></tr><tr><td> A </td><td> CD4CD25 - </td><td> CD4CD25low </td><td>メディアのみ</td><td>メディアのみ</td></tr><tr><td> B </td><td> CD4CD25 - / Tregsの</td><td> CD4CD25low / Tregsの</td><td>のみTregsの</td><td> APCのみ</td></tr><tr><td> C </td><td></td><td></td><td></td><td></td></tr><tr><td> D </td><td></td><td></td><td></td><td></td></tr><tr><td> E </td><td></td><td></td><td></td><td></td></tr><tr><td> F </td><td></td><td></td><td></td><td></td></tr><tr><td> G </td><td></td><td></td><td></td><td></td></tr><tr><td> H </td><td></td><td></td><td></td><td></td></tr></tbody></table>

<ol>
	<li>Barge, A., Cravotto, G., Gianolio, E., Fedeli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+determine+free+Gd+and+free+ligand+in+solution+of+Gd+chelates.+A+technical+note.">How to determine free Gd and free ligand in solution of Gd chelates. A technical note.</a> Contrast Med. Mol. Imaging. 1, 184-188 (2006).</li><li>Nagaraja, T. N., Croxen, R. L., Panda, S., Knight, R. A., Keenan, K. A., Brown, S. L., Fenstermacher, J. D., Ewing, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+arsenazo+III+in+the+preparation+and+characterization+of+an+albumin-linked,+gadolinium-based+macromolecular+magnetic+resonance+contrast+agent.">Application of arsenazo III in the preparation and characterization of an albumin-linked, gadolinium-based macromolecular magnetic resonance contrast agent.</a> J. Neurosci. Methods. 157, 238-245 (2006).</li><li>Supkowski, R. M., Horrocks, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+determination+of+the+number+of+water+molecules,+q,+coordinated+to+europium(III)+ions+in+solution+from+luminescence+decay+lifetimes.">On the determination of the number of water molecules, q, coordinated to europium(III) ions in solution from luminescence decay lifetimes.</a> Inorg. Chim. Acta. 340, 44-48 (2002).</li><li>Menjoge, A. R., Kannan, R. M., Tomalia, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimer-based+drug+and+imaging+conjugates:+design+considerations+for+nanomedical+applications.">Dendrimer-based drug and imaging conjugates: design considerations for nanomedical applications.</a> Drug Discovery Today. 15, 171-185 (2010).</li><li>Que, E. L., Chang, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responsive+magnetic+resonance+imaging+contrast+agents+as+chemical+sensors+for+metals+in+biology+and+medicine.">Responsive magnetic resonance imaging contrast agents as chemical sensors for metals in biology and medicine.</a> Chem. Soc. Rev. 39, 51-60 (2010).</li><li>Uppal, R., Caravan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+probes+for+cardiovascular+MR+imaging.">Targeted probes for cardiovascular MR imaging.</a> Future Med. Chem. 2, 451-470 (2010).</li><li>Major, J. L., Meade, T. J. B. i. o. r. e. s. p. o. n. s. i. v. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioresponsive,+cell-penetrating,+and+multimeric+MR+contrast+agents.">Bioresponsive, cell-penetrating, and multimeric MR contrast agents.</a> Acc. Chem. Res. 42, 893-903 (2009).</li><li>Datta, A., Raymond, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gd-hydroxypyridinone+(HOPO)-based+high-relaxivity+magnetic+resonance+imaging+(MRI)+contrast+agents.">Gd-hydroxypyridinone (HOPO)-based high-relaxivity magnetic resonance imaging (MRI) contrast agents.</a> Acc. Chem. Res. 42, 938-947 (2009).</li><li>Le&#244;n-Rodr&#237;guez, L. M. D., Lubag, A. J. M., Malloy, C. R., Martinez, G. V., Gillies, R. J., Sherry, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responsive+MRI+agents+for+sensing+metabolism+in+vivo.">Responsive MRI agents for sensing metabolism in vivo.</a> Acc. Chem. Res. 42, 948-957 (2009).</li><li>Castelli, D. D., Gianolio, E., Crich, S. G., Terreno, E., Aime, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+containing+nanosized+systems+for+MR-molecular+imaging+applications.">Metal containing nanosized systems for MR-molecular imaging applications.</a> Coord. Chem. Rev. 252, 2424-2443 (2008).</li><li>Caravan, P., Ellison, J. J., McMurry, T. J., Lauffer, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gadolinium(III)+chelates+as+MRI+contrast+agents:+structure,+dynamics,+and+applications.">Gadolinium(III) chelates as MRI contrast agents: structure, dynamics, and applications.</a> Chem. Rev. 99, 2293-2352 (1999).</li><li>Lauffer, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paramagnetic+metal+complexes+as+water+proton+relaxation+agents+for+NMR+imaging:+theory+and+design.">Paramagnetic metal complexes as water proton relaxation agents for NMR imaging: theory and design.</a> Chem. Rev. 87, 901-927 (1987).</li><li>Yoo, B., Pagel, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+responsive+MRI+contrast+agents+for+molecular+imaging.">An overview of responsive MRI contrast agents for molecular imaging.</a> Front. Biosci. 13, 1733-1752 (2008).</li><li>Pandya, S., Yu, J., Parker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+emissive+europium+and+terbium+complexes+for+molecular+imaging+and+sensing.">Engineering emissive europium and terbium complexes for molecular imaging and sensing.</a> Dalton Trans. 23, 2757-2766 (2006).</li><li>Nwe, K., Xu, H., Regino, C. A. S., Bernardo, M., Ileva, L., Riffle, L., Wong, K. J., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+in+the+preparation+of+dendrimer-based+bifunctional+diethylenetriaminepentaacetic+acid+MR+contrast+agent+derivatives.">A new approach in the preparation of dendrimer-based bifunctional diethylenetriaminepentaacetic acid MR contrast agent derivatives.</a> Bioconjugate Chem. 20, 1412-1418 (2009).</li><li>Nwe, K., Bernardo, M., Regino, C. A. S., Williams, M., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+MRI+properties+between+derivatized+DTPA+and+DOTA+gadolinium-dendrimer+conjugates.">Comparison of MRI properties between derivatized DTPA and DOTA gadolinium-dendrimer conjugates.</a> Bioorg. Med. Chem. 18, 5925-5931 (2010).</li><li>Caravan, P., Das, B., Deng, Q., Dumas, S., Jacques, V., Koerner, S. K., Kolodziej, A., Looby, R. J., Sun, W. -. C., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lysine+walk+to+high+relaxivity+collagen-targeted+MRI+contrast+agents.">A lysine walk to high relaxivity collagen-targeted MRI contrast agents.</a> Chem. Commun. , 430-432 (2009).</li><li>Le&#244;n-Rodr&#237;guez, L. M. D., Kovacs, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synthesis+and+chelation+chemistry+of+DOTA-peptide+conjugates.">The synthesis and chelation chemistry of DOTA-peptide conjugates.</a> Bioconjugate Chem. 19, 391-402 (2008).</li><li>Boswell, C. A., Eck, P. K., Regino, C. A. S., Bernardo, M., Wong, K. J., Milenic, D. E., Choyke, P. L., Brechbiel, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+characterization,+and+biological+evaluation+of+integrin+&#945;V&#946;3-targeted+PAMAM+dendrimers.">Synthesis, characterization, and biological evaluation of integrin &#945;V&#946;3-targeted PAMAM dendrimers.</a> Mol. Pharm. 5, 527-539 (2008).</li></ol>

利害の衝突は宣言されません。

Tregsのへの唯一のユニークな特徴として、抑制機能は、同じ内および異なる研究間の疾患の開発のさまざまな段階での被験者の間で確実にかつ均一にテストする必要があります。我々は、このアッセイの標準化への貢献として、当研究室で開発抑制アッセイの詳細を提供します。私たちの広範な最適化の研究では、我々は自然の刺激を提供するAPCとしてPBMCとの併用で抗ヒトCD3でコーティング...

<table border="1"><tbody><tr><th>試薬や器具の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td>をFicoll - Paque PLUS </td><td>アマシャムファルマシアバイオテク</td><td> 17-1440-03 </td><td></td></tr><tr><td> DPBS - 1X </td><td>ギブコ</td><td> 14190-144 </td><td></td></tr><tr><td>トリパンブルー</td><td>インビトロジェン</td><td> 15250-061 </td><td></td></tr><tr><td>抗CD4マイクロビーズ</td><td>国内でのみ有効</td><td> 130-045-101 </td><td></td></tr><tr><td>プレセパレーションフィルター</td><td>国内でのみ有効</td><td> 130-041-407 </td><td></td></tr><tr><td> LSカラム</td><td>国内でのみ有効</td><td> 130-042-401 </td><td></td></tr><tr><td> EDTA </td><td>インビトロジェン</td><td> 15575-020 </td><td></td></tr><tr><td> BSA </td><td>シグマアルドリッチ</td><td> B4287 </td><td></td></tr><tr><td>抗ヒトCD4 - APCCy7（クローンRPA - T4） </td><td> BD Pharmingen社</td><td> 557852 </td><td></td></tr><tr><td>抗ヒトCD25 - PE（クローンM - A251、IL -2Rα） </td><td> BD Pharmingen社</td><td> 555432 </td><td></td></tr><tr><td>抗ヒトCD8 - FITC（クローンRPA - T8） </td><td> BD Pharmingen社</td><td> 555366 </td><td></td></tr><tr><td>抗ヒトCD14 - FITC（クローンM5E2、LPSの受容体） </td><td> BD Pharmingen社</td><td> 555397 </td><td></td></tr><tr><td>抗ヒトCD32 - FITC（クローンFLI8.26、FcγRのタイプII） </td><td> BD Pharmingen社</td><td> 555448 </td><td></td></tr><tr><td>抗ヒトCD116 - FITC（クローンM5D12、GM -CSFRαチェーン） </td><td> BD Pharmingen社</td><td> 554532 </td><td></td></tr><tr><td> Dynalbeads M - 450 tosylactivated </td><td>インビトロジェン</td><td> 140から13 </td><td></td></tr><tr><td>抗ヒトCD3 </td><td> Ancell </td><td> 144から024 </td><td></td></tr><tr><td> Buffer1 </td><td>手作り</td><td></td><td> 0.1MのNa 2 B 4 O 7 pH7.6 </td></tr><tr><td> Buffer2 </td><td>手作り</td><td></td><td> PBS/2mM EDTA / 0.1％BSA pH7.4の</td></tr><tr><td> Buffer3 </td><td>手作り</td><td></td><td> 0.2M Tris/0.1％BSA pH8.5 </td></tr><tr><td>完全RPMI培地</td><td>手作り</td><td></td><td> RPMI 1640培地2mM L -グルタミン、5mMのHEPES 100 U /μg/ mlのpeni / strept 0.5mMのピルビン酸ナトリウム</td></tr><tr><td> [3 H]チミジン</td><td>パーキンエルマー</td><td> NET027Z005MC </td><td></td></tr><tr><td>人間のプールされたAB血清</td><td>アトランタバイオ</td><td> S40110 </td><td></td></tr><tr><td>マルチスクリーン収穫プレート</td><td>ミリポア</td><td> MAHFC1H60 </td><td></td></tr><tr><td> Microscint 20 </td><td>パーキンエルマー</td><td> 6013621 </td><td></td></tr></tbody></table>

human in vitro suppression as screening tool for the recognition of an early state of immune imbalance

制御性T細胞（Treg）は自己抗原に対する免疫寛容の重要なメディエーターである。さらに、彼らは感染後の免疫応答の重要な調節因子である。 Tregsの上でユニークな表面マーカーを同定するための努力にもかかわらず、唯一のユニークな特徴は、エフェクターT細胞の増殖や機能を抑制する能力です。それが唯一のin vitroアッセイにおいてヒトTregの機能を評価に使用される場合があることは明らかですが健康な細胞と細胞（1型糖尿- T1Dのような）自己免疫疾患を有する被験者から分離された横断的研究からの結果を評価する際に、これは問題となる必 ​​要があります。比較する。応答者の比率とのin vitro抑制アッセイにおけるヒトで使用されている抗原提示細胞（APC）の数と種類：レスポンダーT細胞、刺激の性質と強さ、Tregの数と種類の研究所の間で大きなばらつきがあります。この変動は、困難なTregの機能を測定する研究間の比較を行います。 Tregのフィールドには、健常者と自己免疫疾患患者の両方でうまく動作する標準化された抑制アッセイを必要とします。我々は、膵β細胞の自己免疫破壊の根底にあるの被験者に比べて健康なボランティアから単離したT細胞の刺激ではほとんどのイントラアッセイのばらつきを示すin vitroでの抑制アッセイで開発した。この作品の主な目的は、異なる被験者グループ間の比較を可能にするin vitroでヒトの抑制アッセイで記述することです。さらに、このアッセイは、このように予防的免疫療法の1から利益を得ることができる科目を識別する、機能をnTregに小さな損失を描くと、将来のさらなる損失を予測する可能性を秘めています。以下、我々は、この手順に必要な手順の徹底的な説明を提供しています。我々はTregの機能を測定するために使用されるin vitroでの抑制アッセイの標準化に貢献したいと考えています。さらに、我々は免疫不均衡の初期の状態とT1Dのための潜在的な機能のバイオマーカーを認識するツールとして、このアッセイを提供する。

Watch this Scientific Journal Video about Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance at JoVE.com

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregsのは、免疫系の強力な抑制因子である。それ故に、それらを定義するためにユニークな表面マーカーの欠如が存在する、Tregsのの定義は、主に機能的です。ここでは、最適化を説明<em&gt; in vitroで</emT1D開発するリスクのある被験者において免疫不均衡を識別することができる&gt;アッセイ。

ヒトのインビトロ</em免疫バランスの初期状態の認識のためのスクリーニングツールとして>抑制

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

human-vitro-suppression-as-screening-tool-for-recognition-an-early

Research

JoVE Journal

Immunology and Infection

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

14.5K ビュー.  Medical College of Wisconsin. Tregsのは、免疫系の強力な抑制因子である。それ故に、それらを定義するためにユニークな表面マーカーの欠如が存在する、Tregsのの定義は、主に機能的です。ここでは、最適化を説明 in vitroでアッセイ。

ビデオ: Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

初代ヒトナイーブおよびメモリーT細胞から誘導した調節性T細胞の生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

免疫・感染学

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

CD8 による培養腫瘍細胞のアポトーシスのリアルタイム検出+ T 細胞腫瘍浸潤骨髄細胞の免疫抑制機能を勉強するには

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

がん研究

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

抗原特異的規制用のトランス生体内遅延型過敏アッセイ

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

インビトロアッセイで HIV特異的CD4 T細胞エフェクター機能に対する免疫調節経路の影響を評価する

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

臍帯血移植後の水痘帯状疱疹ウイルス特異的細胞性免疫を評価するために、IFN-γELISPOTアッセイの開発

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

アン<em&gt;インビトロ</em&gt; HIV重感染のコンテキストでのマラリアに対する免疫応答を測定するためのモデル

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

TGF-βを含むプロトコルを使用したナイーブCD4 + T細胞からのヒトCD4 + FOXP3 +誘導された調節性T細胞（iTregs）のインビトロ分化における

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

体外と体内の T, B, 骨髄細胞の抑制活性と移植から体液性応答の評価

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

T 細胞受容体を介してライブラリ化学阻害剤のスクリーニングのメディエーターの同定

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Predictive Immune Modeling of Solid Tumors

Immunotherapies show promise in the treatment of oncology patients, but complex heterogeneity of the tumor microenvironment makes predicting treatment response challenging. The ability to resolve the relative populations of immune cells present in and around the tumor tissue has been shown to be clinically-relevant to understanding response, but is limited by traditional techniques such as flow cytometry and immunohistochemistry (IHC), due the large amount of tissue required, lack of accurate cell type markers, and many technical and logistical hurdles. One assay (e.g., the ImmunoPrism Immune Profiling Assay) overcomes these challenges by accommodating both small amounts of RNA and highly degraded RNA, common features of RNA extracted from clinically archived solid tumor tissue. The assay is accessed via a reagent kit and cloud-based informatics that provides an end-to-end quantitative, high-throughput immuno-profiling solution for Illumina sequencing platforms. Researchers start with as few as two sections of formalin-fixed paraffin-embedded (FFPE) tissue or 20-40 ng of total RNA (depending on sample quality), and the protocol generates an immune profile report quantifying eight immune cell types and ten immune escape genes, capturing a complete view of the tumor microenvironment. No additional bioinformatic analysis is required to make use of the resulting data. With the appropriate sample cohorts, the protocol may also be used to identify statistically significant biomarkers within a patient population of interest.

The use of an RNA-based approach to determine quantitative immune profiles of solid tumor tissues and leverage clinical cohorts for immune-oncology biomarker discovery is described through a molecular and informatics protocols.

固形腫瘍の予知免疫モデリング

Immunotherapies show promise in the treatment of oncology patients, but complex heterogeneity of the tumor microenvironment makes predicting treatment ...

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