Name: rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development
Uploaded: 2018-06-19T13:00:00.000+00:00
Duration: 9 min 3 s
Description: Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

私たちの技術的な助手にお世話になっております: u. Bröder、h. Shkarlet 実験手順の中で私たちを助けた。この作品は、部分的 (UniVax、契約番号 601738、TRANSVAC2、契約番号730964) EU 補助金及びヘルムホルツ協会助成金 (ハイ-IDR) によって賄われていた。資金源に影響しなかった研究デザイン、原稿や文書の提出する決定の世代。

本研究で使用されるすべてのマウスは、C57BL/6 背景からあった。すべての動物は、病原体フリーの条件の下で保たれました。(TierSchG BGBl ドイツの動物保護法の規範に従ってすべての実験が行われました。私 S 1105;25.05.1998) 許可番号 33.4-42502-04-13/1281 と 162280 の下で動物実験の倫理および州のオフィス (低いザクセン州事務所の消費者保護、食の安全) 低いザクセン州委員会によって承認されました。
1. CFSE 染色 OT-私は T 細胞と養子の転送
注: OT-私マウス固定 α と β 鎖一緒に OVA の免疫支配的なペプチドを認識する T 細胞受容体 (TCR) を表す transgenically 生成された動物は、SIINFEKL10,11。その結果、これらのマウスはかなり高い SIINFEKL 固有 CD8 数を持っている+ T 細胞 (97%)12通常または OVA 接種マウス (≤ 1%)13と比較した場合。
<ol><li>トレーサブルな CD8 の分離 OT から+ T 細胞-私 T リンパ球特定Thy1 (Thy1.1) の対立遺伝子を発現するマウス。<ol>
<li>6-9 週齢の OT を安楽死させる-私 CO2吸入によるマウスに続いて頚部転位14。手術ペンチや鉗子の助けを借りて体から皮膚を切り離す手術のハサミで皮膚を切断することによって脾臓および主要なリンパ節 (鼠径部、腋窩、頸部ペアのみ) を分析し、各を含むペトリ皿 (60 x 15 mm) に配置、100 μ m 孔メッシュ カップ組織研削やセル リリース。</li>
		<li>3-5 mL の完全 RPMI 培 (RPMI 1640 年 10 %v/v FCS、100 U/mL ペニシリン、ストレプトマイシン 50 μ G/ml) 氷で保たれるペトリ皿 (それぞれ 1 つのメッシュ カップ) で維持されます。</li>
		<li>シリンジのプランジャーまたは同様の滅菌器の助けを借りて、臓器をマッシュ (前の切断は必要ありません) 15 mL 遠沈管の結果単一細胞懸濁液を収集し、。</li>
		<li>4 ° C で 10 分間 300 x g で細胞懸濁液を遠心分離します。上清を捨てます。遠心分離で 10 mL の冷 PBS のセルを洗浄し、塩化アンモニウムのバッファー (ACK バッファー、市販) の 1 mL/脾臓にペレットを再懸濁による脾臓から赤血球を溶解させます。氷の上 1.5 分細胞をインキュベートし、その後 (前に、と) は遠心分離によって 10 ml の冷 PBS のセルを洗ってください。</li>
		<li>再 1 ml の PBS (pH 7.2) 含む 5% ウシ胎児血清磁気分離のための同じ管でペレットを中断します。</li>
	</ol>
</li>
	<li>磁気分離を実行する CD8 の負の淘汰に進みます+ T の製造元の指示に従って磁気分離キットを使用してセルまたはプロトコル15を公開します。</li>
	<li>CFSE 染色するには、まずカウント CD8 の数+ T OT から得られた細胞-私自動化された細胞カウンター (パーティクル カウンター) を使用してマウス。1-5 107セル/mL 37 ° C、15 ml の隼の管の光から保護で 7 分の 5 μ m CFSE PBS で 1-5 mL の量 x を染色します。</li>
	<li>牛胎児血清の同じボリューム (1:1) をセルに追加することによって CFSE 染色を癒やす、光から保護されている 37 ° C でさらに 7 分間インキュベートするそれら。10 ml の PBS のセルを 2 回洗います。</li>
	<li>自動化された細胞カウンターを使用してセルをカウントします。3-5 x の 100 μ L の 10 の6セル/マウスを静脈内に注入するために 3-5 x 107セル/mL の PBS のセル番号を設定 (静注) 尾静脈を介して。</li>
	<li>尾静脈注射を実行するには、適切な restrainers でマウスを固定します。背部区域と尾静脈血管拡張を許可する 1 〜 3 分のマウスから 20 〜 25 cm の間置かれた赤い光ランプを使用して注入するマウスの尾を温めます。 注: これは簡単静脈検出と細胞懸濁液の管理に役立ちます。</li>
	<li>(マウスとランプの間に置かれた手) でもマウスのホットし、尾静脈がはっきりと見える、インジェクションに進むではないです。25 ゲージ針161 mL 注射器を使用して水平または背の尾静脈にセルを挿入します。</li>
</ol>2. 予防接種 (補助 + 遠藤無料 OVA)
<ol><li>OT を移植されたマウスを置く-私は麻酔室で (ステップ 1.7、図 1) を細胞し、酸素のイソフルラン麻酔マシン14管理します。</li>
	<li>マウスを麻酔下で完全に眠っているとき、チャンバーから取り出すし、臀筋浅(外側腰部) にきれいな注入を行うために電気髪のナイフを使用して領域のマウスの毛皮を剃るアプリケーション領域の景色の良い。</li>
	<li>ワクチン、25 ゲージ針を使用して剃毛エリアでサウスカロライナの 50 μ L を注入します。</li>
</ol>3 フローサイトメトリー解析のための汚損およびリンパ球の分離
<ol><li>脾細胞およびリンパ球の分離<ol>
<li>頚部転位続いて CO2吸入ワクチン接種マウスを安楽死させます。</li>
		<li>排水、遠くのリンパ節や脾臓を抽出し、100 μ m 孔メッシュ カップ組織研削と細胞のリリースを含む別のペトリ皿に配置します。1.1.2 1.1.3 からの手順に従います。</li>
		<li>遠心分離後、上清をデカントし、再残骸 PBS の同じボリューム (約 100 μ L) の細胞ペレットを中断します。</li>
	</ol>
</li>
	<li>フローサイトメトリー解析のため染色<ol>
<li>15 mL 遠心管中の表 1に示されている濃度で染色抗体を含むマスター ミックスを調製、染色ミックスを集中してこのように 2 倍を降伏します。サンプルあたり 100 μ L を持っているマスター ミックスの十分な量を準備します。セルと 1:1 のマスターの組合せをミックスし、4 の ° c で 30 分間インキュベートします。 注: サンプルあたり最終染色巻 200 μ L (抗体のマスターの組合せの細胞ペレット 100 μ L の 100 μ L) をする必要があります。</li>
		<li>1.1.3、10 mL の PBS を 2 回追加するで説明するよう 2 回遠心分離によって細胞を洗ってください。</li>
		<li>0.5 - 1 染色細胞を再停止集録および流れの cytometer チューブへの転送停止用 mL の PBS。常に氷の上のサンプルを維持し、光から保護されています。</li>
	</ol>
</li>
</ol>4. フローサイトメトリー
<ol><li>常に 70-100 μ m のフィルターを使用してセルのサンプルがプレフィルターです。</li>
	<li>表 1 (ビーズまたは細胞) に詳細な fluorophores の単一の染色補正コントロールを準備します。 注: 補償する必要がありますの cytometer または分析ソフトウェア17,18,19で実行し、すべてのサンプルに適用されます。</li>
	<li>図 2にゲートの戦略に従います。前方散乱領域 (図 2 a)、もう一度広い側の散布面積 (SSA は、図 2 b) ダブレットを除外するために対側方散乱と前方散乱の高さで簡単にゲート集団。</li>
	<li>門真 CFSE を差別するために BV 650 (自動蛍光) FITC (CFSE) 対をプロットすることによって図 2 bから人口は高自動蛍光細胞から細胞を染色しました。ビーズや補正コントロールに BV 650 に共役抗体で染色のセルが含まれます。OT を門に FITC 対 BV 650 のこのプロット (図 2) を使用-私 CFSE は細胞を染色します。</li>
	<li>受信者マウス対ドナー由来細胞を区別するために Pe Cy7 (Thy1.1 - CD90.1-) を APC (CD8) 対の図 2 Cから人口をゲートします。 注: セルに由来する OT-私 WT (C57BL/6、受信者) マウス由来細胞は Thy1.2+ (CD90.2) (図 2 D) に対し、ドナーのマウスは Thy1.1+ (CD90.1)。いくつかの研究所がある OT-私 Thy1.2 バック グラウンドで WT (C57BL/6)、Thy1.1 のマウス。この場合は OT の Thy1.2 抗体を使用する必要が-ゲート携帯ですね。</li>
	<li>CD8 を再ゲート+細胞 Thy1.1+人口 APC (CD8) 対 BV 450 (CD4) で構成されるゲートにプロットして (図 2 e)。</li>
	<li>CD8 のヒストグラム表示を使用して、図2 e ゲート人口を表示+ CFSE (FITC、530/15 チャンネル - 青色レーザー-)、細胞増殖の人口門 10 から強度を含む2レベルまで、コントロール分裂していない人口は (強度 10 〜5、CFSE 染色と流れの cytometer の電圧設定の有効性に応じて) (図 2 f)。</li>
	<li>マーカーにする、(図 2 には表示されません) 付加ゲート プロット FITC 対 L/D (450/20 チャンネル、紫外レーザー) 増殖の評価に並列に死んだ細胞を評価するために。</li>
	<li>補正コントロールのイベントの少なくとも 5000 と流れの cytometer でサンプルから 10.000 イベント (図 2E、ゲート) を取得します。低または中程度の流れの cytometer を実行 (20.000 イベント/秒の流量を超えない)。</li>
</ol>

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American journal of physiology.</a> Lung cellular and molecular physiology. , L54-L63 (2012).</li><li>Burgdorf, S., Scholz, C., Kautz, A., Tampe, R., Kurts, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+mechanistic+separation+of+cross-presentation+and+endogenous+antigen+presentation.">Spatial and mechanistic separation of cross-presentation and endogenous antigen presentation.</a> Nature. 9, 558-566 (2008).</li><li>Last'ovicka, J., Budinsky, V., Spisek, R., Bartunkova, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+lymphocyte+proliferation:+CFSE+kills+dividing+cells+and+modulates+expression+of+activation+markers.">Assessment of lymphocyte proliferation: CFSE kills dividing cells and modulates expression of activation markers.</a> Cellular immunology. , 79-85 (2009).</li><li>Oelke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+CD8(+)+antigen-specific+cytotoxic+T+lymphocytes+after+enrichment+based+on+cytokine+secretion:+comparison+with+the+MHC-tetramer+technology.">Functional characterization of CD8(+) antigen-specific cytotoxic T lymphocytes after enrichment based on cytokine secretion: comparison with the MHC-tetramer technology.</a> Scand J Immunol. 52, 544-549 (2000).</li><li>Wang, W., Golding, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytotoxic+T+lymphocyte+response+against+a+protein+antigen+does+not+decrease+the+antibody+response+to+that+antigen+although+antigen-pulsed+B+cells+can+be+targets.">The cytotoxic T lymphocyte response against a protein antigen does not decrease the antibody response to that antigen although antigen-pulsed B cells can be targets.</a> Immunology letters. 100, 195-201 (2005).</li><li>O'Sullivan, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+CD8(+)+T+cells+use+cell-intrinsic+lipolysis+to+support+the+metabolic+programming+necessary+for+development.">Memory CD8(+) T cells use cell-intrinsic lipolysis to support the metabolic programming necessary for development.</a> Immunity. 41, 75-88 (2014).</li><li>Xu, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+interferon+protects+antiviral+CD8++T+cells+from+NK+cell+cytotoxicity.">Type I interferon protects antiviral CD8+ T cells from NK cell cytotoxicity.</a> Immunity. 40, 949-960 (2014).</li><li>Volk, A., et al. <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+three+humanized+mouse+models+for+adoptive+T+cell+transfer.">Comparison of three humanized mouse models for adoptive T cell transfer.</a> The journal of gene medicine. 14, 540-548 (2012).</li><li>Safinia, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+Mice+as+Preclinical+Models+in+Transplantation.">Humanized Mice as Preclinical Models in Transplantation.</a> Methods Mol Biol. 1371, 177-196 (2016).</li><li>Grover, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+NOG+mice+as+a+model+for+tuberculosis+vaccine-induced+immunity:+a+comparative+analysis+with+the+mouse+and+guinea+pig+models+of+tuberculosis.">Humanized NOG mice as a model for tuberculosis vaccine-induced immunity: a comparative analysis with the mouse and guinea pig models of tuberculosis.</a> Immunology. 152, 150-162 (2017).</li></ol>

著者が明らかに何もありません。

現代のワクチンは、理想的にしまいこむ精製抗原とアジュバント、リポソーム、ウイルス様粒子、ナノ粒子やライブのベクトルのような配信システムの可能な追加です。ワクチンを設計するときの重要な側面は、臨床医学のニーズによると右の補助を選択することです。範囲の一部は好む細胞性免疫応答 (またはその両方) と体液性、全身性免疫反応 (またはその両方)、対ローカルの選挙とタ...

細胞傷害性 T リンパ球 (CTL) を誘導するワクチンは、がん1の特定の種類を戦うために開発されているキーの治療です。CTL はまた細胞内病原体2に対する予防ワクチンのため重要です。また、CTL は、初期の生活の感染症5と戦うための CTL はまた決まる人新生児3,4などリスク集団の機能的アクティブないくつかの免疫防御機構の 1 つです。この点では、CTL 応答 (ミョウバン) は発生しませんするアジュバント開発された呼吸器合胞体ウイルス (RSV) に対するワクチンは乳児6感染症時に重篤な合併症につながるワクチンの完全な失敗に終わった。予防接種のこれらの負の影響は CD8 によって反転できます+ T 細胞応答7。我々 は以前、インターフェロン遺伝子 (スティング) 作動薬のいくつかの刺激によって誘発される (タイプ I のインターフェロン) 主なサイトカインの増殖を測定することによって部分的にこれらのアジェバントの8、によって生成された CTL 応答のため不可欠であることを示しています。OT-T 細胞ワクチン接種と CTL 誘導機能の測定で観測されたこれらの結果を使用して拡張予防接種スケジュール9後。OT の拡散の測定-私は CD8+ T 細胞の野生型 (WT) C57BL/6 受信者マウス carboxyfluorescein 染色エステル (CFSE) 色素希釈が生成するワクチンのアジュバントの機能の推定SIINFEKL (OVA 卵白アルブミンの免疫支配的なペプチド) のクロス プライミング。この手法のバリエーションが広く OT の増殖の評価-私は CD8+と OT II CD4+ T 細胞。たとえば、それはいない選択したサイトカイン (KO マウス)、あるいは WT 動物抗原リコール後ワクチンの有効性を測定するために使用されています。CFSE 染色 OT の受身伝達後、短いプロトコル (4 日間実験) を考案-私は CD8+ T 細胞、50 の 1 つの線量から成る皮下皮下接種試験アジュバントを添加したエンドトキシン OVA の μ g を投与(図 1)。フォロー アップの結果ワクチン接種後 48 時間は、CTL 応答を生成するアジュバントの能力の信頼性の高い証拠を提供します。この戦略によって脾臓 (または遠隔リンパ節) に CTL 活性を測定することによって応答の範囲と同様、予防接種後排出リンパ節における局所免疫応答の効力を評価することが可能です。

<table><tbody><tr><td>BD LSR Fortessa Cell Analyzer</td><td>BD</td><td>Special Order</td><td>Flow Cytometer</td></tr><tr><td>CFSE</td><td>Molecular Probes</td><td>C34554</td><td>Proliferation Dye</td></tr><tr><td>MojoSort Mouse CD8 T Cell Isolation Kit</td><td>Biolegend</td><td>480007</td><td>Magnetic Isolation Beads and antibodies for negative selection of untouched CD8 T cells.</td></tr><tr><td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation</td><td>Molecular Probes</td><td>L23105</td><td>Dead Cell Marker</td></tr><tr><td>CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), PE-Cyanine7</td><td>eBioscience</td><td>25-0900-82</td><td>antibody</td></tr><tr><td>APC anti-mouse CD8a Antibody</td><td>BioLegend</td><td>100712</td><td>antibody</td></tr><tr><td>BV421 Rat Anti-Mouse CD4</td><td>BD</td><td>740007</td><td>antibody</td></tr><tr><td>Z2 coulter Particle count and Size Analyzer</td><td>Beckman Coulter</td><td>9914591DA</td><td>Cell counter. Z2 Automated particle/cell counter</td></tr><tr><td>EndoGrade Ovalbumin (10 mg)</td><td>Hyglos(Germany)</td><td>321000</td><td>Ovalbumin endotoxin free tested.</td></tr><tr><td>Cell Strainer 100&amp;micro;m nylon</td><td>Corning</td><td>352360</td><td>Cell strainer (100 &amp;micro;m pore mesh cups).</td></tr><tr><td>Sample Vials</td><td>Beckman Coulter</td><td>899366014</td><td>Sample vials for Z2 automated counter</td></tr><tr><td>C57BL/6 mice (CD90.2)</td><td>Harlan (Rossdorf, Germany)</td><td></td><td>Company is now Envigo</td></tr><tr><td>OT-I (C57BL/6 background, CD90.1)</td><td>Harlan (Rossdorf, Germany)</td><td></td><td>Inbreed at our animal facility. Company from where adquired is now Envigo</td></tr><tr><td>FACS tubes</td><td>Fischer (Corning)</td><td>14-959-5</td><td>Corning Falcon Round-Bottom Polystyrene Tubes</td></tr><tr><td>Falcon 15 mL tubes</td><td>Fischer (Corning)</td><td>05-527-90</td><td>Falcon 15mL Conical Centrifuge Tubes</td></tr><tr><td>PBS (500 mL)</td><td>Fischer (Gibco)</td><td>20-012-027</td><td>Gibco PBS (Phosphate Buffered Saline), pH 7.2</td></tr><tr><td>Red lamp (heating lamp)</td><td>Dirk Rossmann GmbH (Germany)</td><td>405096</td><td>Heating infrred lamp (100 wats)</td></tr><tr><td>IsoFlo (Isoflurane)</td><td>Abbott Laboratories (USA)</td><td>5260.04-05.</td><td>Isoflurane anesthesic (250 mL flask).</td></tr><tr><td>&lt;strong&gt;Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber&lt;/strong&gt;</td><td>Parkland Scientific</td><td>V3000PK</td><td>Isoflurane anesthesia machine.</td></tr><tr><td>RPMI 1640 medium</td><td>Gibco (distributed by ThermoFischer)</td><td>11-875-093</td><td>Base medium with Glutamine (500 mL)</td></tr><tr><td>Pen-Strept antibiotic solution (Gibco)</td><td>Gibco (distributed by ThermoFischer)</td><td>15-140-148</td><td>Gibco Penicillin-Streptomycin (10,000 U/mL)</td></tr><tr><td>Fetal Bobine Serum (Gibco)</td><td>Gibco (distributed by ThermoFischer)</td><td>10082147</td><td>Fetal Bovine Serum, certified, heat inactivated, US origin</td></tr><tr><td>ACK Lysing Buffer (100 ml)</td><td>Gibco (distributed by ThermoFischer)</td><td>A1049201</td><td>Amonium Chloride Potasium (ACK) Whole Blood Lysis Buffer, suitable for erytrocyte lysis in spleen suspensions also</td></tr><tr><td>Plastic Petri Dishes</td><td>Nunc (distributed by ThermoFischer)</td><td>150340</td><td>60 x 15mm Plastic Petri Dish, Non-treated</td></tr><tr><td>Cell Clump Filter</td><td>CellTrics (Sysmex)</td><td>04-004-2317</td><td>CellTrics&amp;reg; 50 &amp;mu;m, sterile</td></tr></tbody></table>

rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development

モダンなサブユニット ワクチンの評価では、中和抗体の生成は重要ですがアジュバントの選択は不十分であることを明らかにします。したがって、細胞傷害性 T リンパ球 (CTL) の反応を促進することができる体液性および細胞免疫促進機能を備えたアジュバントが急務します。したがって、忠実な監視クロスプライミングを誘導し、その CTL 世代を強化補助候補者のワクチンの開発の重要なステップを表します。ここで提案する SIINFEKL 固有を使用するメソッドのアプリケーション (OT-私) T 細胞モデル抗原卵白アルブミン (OVA) は、生体内で異なる補助候補の存在下でのクロス プレゼンテーションを監視します。このメソッドは、最高のクロス プライミング機能を備えたアジュバントを選択する急速なテストを表します。CD8 の増殖+ T 細胞クロスプライミングの最も貴重な徴候、また、アジュバント クロス プレゼンテーションの相互関係として考えています。この機能は、リンパ節や脾臓のようなさまざまな免疫器官で評価できます。CTL 世代の範囲も監視できます、それによりローカルの性質に洞察を与えて (主にリンパ節をドレイン) または全身性反応 (遠くのリンパ節や脾臓)。さらにこの手法も従来型と遺伝子組み換えマウスの系統に使用される可能性を提供していますクロス プレゼンテーションの特定の経路を阻害することができます薬をテストするための複数の変更をことができます。要約すると、ここで紹介するアプリケーションは業界や学界でワクチンの研究所で、開発または化学のアジュバント ワクチンの研究と開発のための変更のため役に立つでしょう。

CTL の発電容量をに対して転送 OT の増殖を測定することによって評価我々 異なるアジュバント (その ADJ1 および ADJ2) の組み合わせを使用して治療法をテストするために-私は CD8+ T 細胞のフローサイトメトリー (図 2)。このため、以前ドレイン リンパ節および脾臓 (表 1) から分離された細胞を染色しました。CD8 の増殖を測定?...

Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

標準的な免疫学的技術のアプリケーションをご紹介 (CFSE 染色 OT-私増殖) 急速に補助を介した細胞傷害性 T リンパ球 (CTL) の生成をモニターするつもり体内。CTL 容量の高速推定、がん治療用ワクチンと同様に、細胞内の病原体に対する予防ワクチンの開発に役立つ。

急速なIn Vivo評価補助の細胞傷害性 T リンパ球生成機能のワクチン開発のため

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

rapid-vivo-assessment-adjuvant-s-cytotoxic-t-lymphocytes-generation

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JoVE Journal

Medicine

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

8.6K ビュー.  Helmholtz Centre for Infection Research. 標準的な免疫学的技術のアプリケーションをご紹介 (CFSE 染色 OT-私増殖) 急速に補助を介した細胞傷害性 T リンパ球 (CTL) の生成をモニターするつもり体内。CTL 容量の高速推定、がん治療用ワクチンと同様に、細胞内の病原体に対する予防ワクチンの開発に役立つ。

ビデオ: Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

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 ...

免疫・感染学

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

T細胞応答の評価のための蛍光ターゲット配列の使用<em&gt;インビボ</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

ナノ粒子ワクチン接種後の抗原特異的CD8 + T細胞応答の分析のための全動物イメージングとフローサイトメトリー技術

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

生体内で予防接種モデルを用いた抗原特異的 T 細胞細胞傷害性関数の検出のための試金

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we investigated immunological effects of a novel lipid-DNA immunostimulant in nanoparticle form from different administration routes in the mouse: oral, intranasal, subcutaneous, footpad, intraperitoneal, and intravenous. These injections will directly influence the immune response, and harvesting lymphatic tissues and analysis of dendritic cell (DC) activation in the tissues are crucial parts of these evaluations. The extraction of mediastinal lymph nodes (mLNs) is important but quite complex because of the size and location of this organ. A stepwise procedure for harvesting the inguinal lymph node (iLN), mLN, and spleen and analyzing DC activation by flow cytometry is described.

Experimental procedures for the subsequent extraction of lymphatic tissues to test lymphoid dendritic cell activation are described after treatment of an immunostimulating nanomaterial.

免疫活性化エージェント評価: リンパ組織抽出・注入経路依存性樹状細胞活性化

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

腫瘍特異的 CD8+ T 細胞応答における Immunodominance を研究するためのインビボ細胞毒性アッセイ

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; in particular, some MHC I-restricted epitopes can induce effective cytotoxic T lymphocyte activity to clear tumor cells. Additionally, nasal administration is an effective and safe delivery technique for tumor vaccines due to its convenience and improved patient compliance. However, epitope peptides are unsuitable for nasal delivery because of their poor immunogenicity and lack of delivery efficiency. Nanoemulsions (NEs) are thermodynamically stable systems that can be loaded with antigens and delivered directly to the nasal mucosal surface. Ile-Lys-Val-Ala-Val (IKVAV) is the core pentapeptide of laminin, an integrin-binding peptide expressed by human respiratory epithelial cells. In this study, an intranasal self-assembled epitope peptide NE tumor vaccine containing the synthetic peptide IKVAV-OVA257-264 (I-OVA) was prepared by a low-energy emulsification method. The combination of IKVAV and OVA257-264 can enhance antigen uptake by nasal mucosal epithelial cells. Here, we establish a protocol to study the physicochemical characteristics by transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS); stability in the presence of mucin protein; toxicity by examining the cell viability of BEAS-2B cells and the nasal and lung tissues of C57BL/6 mice; cellular uptake by confocal laser scanning microscopy (CLSM); release profiles by imaging small animals in vivo; and the protective and therapeutic effect of the vaccine by using an E.G7 tumor-bearing model. We anticipate that the protocol will provide technical and theoretical clues for the future development of novel T cell epitope peptide mucosal vaccines.

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo.

インビトロおよびインビボでの鼻自己組織化ナノエマルジョン腫瘍ワクチンの調製、特性、毒性、および有効性評価

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; ...

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

インビトロ ナノエマルジョンワクチンアジュバントオフィオポゴニンDの細胞活性評価

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

ウシ単球由来樹状細胞を用いたワクチン免疫原性の決定

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...

An Immunological Technique to Monitor Adjuvant-Mediated Cytotoxic T Lymphocyte Generation

Source: Lirussi, D. et al., <a href="https://app.jove.com/v/57401">Rapid In Vivo&#160;Assessment of Adjuvant&#39;s Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development.</a> J. Vis. Exp. (2018)
The video demonstrates an immunological method to monitor in vivo adjuvant-induced cytotoxic T lymphocyte or CTL production. A mouse is pre-injected with genetically engineered, proliferative dye-stained donor CD8+ T lymphocytes. Subcutaneous injection of ovalbumin with adjuvants generates cytotoxic CD8+ T lymphocytes that are identified post-harvesting through immunostaining and flow cytometry.

アジュバント媒介性細胞傷害性Tリンパ球の生成をモニターする免疫学的手法

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
All mice ...

急速なIn Vivo評価補助の細胞傷害性 T リンパ球生成機能のワクチン開発のため

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