Name: rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development
Uploaded: 2018-06-19T13:00:00
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?...

<ol>
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F., Berger, H., Su, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Activation+of+Murine+Lymphocytes.">Isolation and Activation of Murine Lymphocytes.</a> Journal of visualized experiments: JoVE. , (2016).</li><li>Shimizu, S., Bullock, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Laboratory Mouse. , 527-542 (2004).</li><li>Breton, G., Lee, J., Liu, K., Nussenzweig, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+human+dendritic+cell+progenitors+by+multiparametric+flow+cytometry.">Defining human dendritic cell progenitors by multiparametric flow cytometry.</a> Nature protocols. 10, 1407-1422 (2015).</li><li>Kaminski, D. A., Wei, C., Rosenberg, A. F., Lee, F. E. -. H., Sanz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparameter+Flow+Cytometry+and+Bioanalytics+for+B+Cell+Profiling+in+Systemic+Lupus+Erythematosus.">Multiparameter Flow Cytometry and Bioanalytics for B Cell Profiling in Systemic Lupus Erythematosus.</a> Methods in molecular biology. 900, 109-134 (2012).</li><li>Bayer, J., Grunwald, D., Lambert, C., Mayol, J. F., Maynadie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thematic+workshop+on+fluorescence+compensation+settings+in+multicolor+flow+cytometry.">Thematic workshop on fluorescence compensation settings in multicolor flow cytometry.</a> Cytometry. Part B, Clinical cytometry. 72, 8-13 (2007).</li><li>Newrzela, S., 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=T-cell+receptor+diversity+prevents+T-cell+lymphoma+development.">T-cell receptor diversity prevents T-cell lymphoma development.</a> Leukemia. 26, 2499-2507 (2012).</li><li>Iwasaki, 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=Allergen+endotoxins+induce+T-cell-dependent+and+non-IgE-mediated+nasal+hypersensitivity+in+mice.">Allergen endotoxins induce T-cell-dependent and non-IgE-mediated nasal hypersensitivity in mice.</a> J Allergy Clin Immunol. 139, 258-268 (2017).</li><li>Tsuchiya, K., Siddiqui, S., Risse, P. A., Hirota, N., Martin, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+presence+of+LPS+in+OVA+inhalations+affects+airway+inflammation+and+AHR+but+not+remodeling+in+a+rodent+model+of+asthma.+American+journal+of+physiology.">The presence of LPS in OVA inhalations affects airway inflammation and AHR but not remodeling in a rodent model of asthma. 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>

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&#181;m nylon</td>
			<td>Corning</td>
			<td>352360</td>
			<td>Cell strainer (100 &#181;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>Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber</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&#174; 50 &#956;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. ...

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Medicine

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

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Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

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