この作品は、NIHのR01 CA104757グラント（MH Manjili）によってサポートされていました。我々は感謝VCUマッシーがんセンターの支援とがん研究のための連邦基金を認めます。

1。リンパ球5の単離<ol><li>担癌FVBN202トランスジェニックマウスからの腫瘍所属リンパ節または脾臓を分離し、10％FBSを添加した氷冷RPMI1640で単一の細胞懸濁液を準備する。ポリスチレンチューブに比べて大きく、T細胞の収率で50 mlのポリプロピレンコニカルチューブの結果でB / I活性化。ケタミンとキシラジンの麻酔のための腹腔内注射されています。頸椎脱臼は、安楽死の方法として使用されます。 </li><li>文化16時間のIL - 2（Peprotech社製）の80 U / mLのと一緒にブリオスタチン- 1（5 nM）およびイオノマイシン（1μM）で、15％FBSを含む完全培地中の細胞（10 6細胞/ mL） </li><li> 24時間のIL - 7（10 ng / ml）およびIL - 15（10 ng / mL）を（Peprotech社製）との完全培地に10 6細胞/ mLの暖かい培地（37℃）および培養で細胞を3回洗浄。 </li><li> 24時間のためのIL - 2（40 U / mL）で細胞をパルス</li><li>さらに4日間、細胞と培養してIL - 7およびIL - 15（10 ng / mL）を持つを分割する。培地を変更し、2日ごとに必要に応じてセルを分割。 </li></ol> 2。細胞数とフローサイトメトリー分析5によってT細胞のフォールド展開を決定する<ol><li>光顕微鏡による細胞の数<ol><li>トリパンブルーで適切な細胞の希釈（1:100）を準備し、血球計算盤の上にいくつかμLを追加</li><li> 9の正方形をカウントし、希釈率を掛けた室の数に細胞数を割ることによって、総細胞数を決定します。結果は数× 10 4細胞/ mLを紹介します。 </li></ol></li><li>フローサイトメトリーによる拡張セルにCD8 +およびCD4 + T細胞の割合を決定する<ol><li>培養することにより、Fc受容体に対する抗体の非特異的結合氷上で20分間anti-CD16/CD32抗体（Biolegend）で細胞をブロックし、1％のアジ化ナトリウムを添加した氷冷PBS 2mLで細胞を2回洗浄。 </li><li>氷上で20分間、FITC - CD4およびPE - CD8抗体で培養することによって細胞を染色し、1％FBSおよび0.1％アジ化ナトリウムを添加した氷冷PBS 2mLで細胞を2回洗浄する。 </li><li> 1％パラホルムアルデヒドで細胞を固定し、ベックマンコールター社FC 500でサンプルを実行し、サミットのバージョン4.3ソフトウェアを使用して分析する。 </li></ol></li></ol> 3。 ex vivoでの拡張性T細胞の腫瘍特異性を決定する<ol><li>文化24時間5の照射神経正MMCの腫瘍細胞（15,000ラジアン）で10:1の比率で完全培地での生体外で展開されたリンパ球</li><li>使用° Cまで-80℃で収穫の上清とストア。5,6 </li><li>検出し、IFN -γ、製造元のプロトコールに従って、マウスIFN -γELISAセット（BD Pharmingen社製）を使用して。5,6 </li></ol> 4。 5,6 ex vivoでの拡張性T細胞の抗腫瘍機能を決定する<ol><li> 3 mLの完全培地（グルタミンおよびβペニシリンの100U / mLを、100μg/ mlのストレプトマイシン、10％FBS、を補充したRPMI - 1640で完全培地で48時間のための標的比10:1のエフェクターの腫瘍細胞とT細胞をインキュベート-メルカプトエタノール）と6ウェル培養皿でのIL - 2の20U / mLの（Peprotech社製）37℃/ 5％CO 2。 </li><li>製造業者のプロトコルに従って、PE -抗マウスIgG、アネキシンV - FITCとヨウ化プロピジウム（PI）（BD Pharmingen社）に続いて神経の3つのカラー抗体染色（anti-c-Erb2/c-neu、クローンA - 4、カルビオケム）を実行</li><li>腫瘍細胞の神経陽性腫瘍細胞上にゲートと分析可能性（アネキシンV-/PI-） </li></ol> 5。乳癌のマウスモデル FVBN202トランスジェニック雌マウス（Charles River Laboratories）は、腫瘍反応性T細胞の源に使用することができます。これらのマウスは、MMTVプロモーターの制御下でアクティブになっていないラット神経遺伝子を過剰発現し、その結果として、7歳の40〜10カ月の間に自発的な乳癌を開発する。これらのマウスは自発carcinoma8の開発前に非浸潤性乳管癌（DCIS）と同様の前癌性乳腺肥大症を開発する。自然発生腫瘍を有するマウスは、T細胞のドナーとして使用されています。 6。代表的な結果： in vivoでの腫瘍で感作されていないナイーブT細胞の殺傷に16時間の結果を得るためにB / IとT細胞の活性化。腫瘍反応性T細胞のB / I選択した後、彼らは、γ鎖のサイトカイン（図1）6日間の文化の中で2.8倍にまで拡大する。両方CD8 +およびCD4 + T細胞が均等にγ鎖のサイトカイン（図2）で展開されます。 エキソビボ -拡張されたT細胞は、神経正のマウス乳腺癌（MMC）腫瘍細胞（図3）の存在下でIFN -γの産生により評価としてドナーマウスは、に感作されたこと腫瘍に対する高い応答性を示す。 T細胞を拡大し、ex vivoでの神経正MMCの腫瘍のセルにアポトーシスを誘導することができるlsの腫瘍細胞のように生存率は（図4）48時間以内に92％から61％に低下します。 <img src="/files/ftp_upload/2381/2381fig1.jpg" alt="欠落している図" /> 図1 B / I活性化（1日目）およびγ鎖のサイトカインとex vivoでの展開（3日目、5、7）以下の異なる時点でのリンパ球のフォールド拡大<img src="/files/ftp_upload/2381/2381fig2.jpg" alt="欠落している図" /> 図2。CD4 +およびCD8 + T細胞の合計パーセンテージ前とγ鎖のサイトカインとの7日間の膨張の後に。 <img src="/files/ftp_upload/2381/2381fig3.jpg" alt="欠落している図" /> 図3。IFN -γを使用して前のγ鎖のサイトカインとの7日間の膨張へと後の担癌マウスから単離したT細胞による産生、IFN -γELISA腫瘍で刺激した<img src="/files/ftp_upload/2381/2381fig4.jpg" alt="欠落している図" /> 図4。神経正のマウス乳腺癌（MMC）の腫瘍細胞に対するガンマ鎖のサイトカインで、T細胞を拡大し、ex vivoでの細胞傷害機能

<ol>
	<li>Goodell, V., Waisman, J., Salazar, L. G., de la Rosa, C., Link, J., Coveler, A. L., Childs, J. S., Fintak, P. A., Higgins, D. M., Disis, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Level+of+HER-2/neu+protein+expression+in+breast+cancer+may+affect+the+development+of+endogenous+HER-2/neu-specific+immunity.">Level of HER-2/neu protein expression in breast cancer may affect the development of endogenous HER-2/neu-specific immunity.</a> Mol Cancer Ther. 7, 449-454 (2008).</li><li>Disis, M. L., Knutson, K. L., Schiffman, K., Rinn, K., McNeel, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-existent+immunity+to+the+HER-2/neu+oncogenic+protein+in+patients+with+HER-2/neu+overexpressing+breast+and+ovarian+cancer.">Pre-existent immunity to the HER-2/neu oncogenic protein in patients with HER-2/neu overexpressing breast and ovarian cancer.</a> Breast Cancer Res Treat. 62, 245-252 (2000).</li><li>Liu, S., Riley, J., Rosenberg, S., Parkhurst, M. <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+common+gamma-chain+cytokines,+interleukin-2,+interleukin-7,+and+interleukin-15+for+the+in+vitro+generation+of+human+tumor-reactive+T+lymphocytes+for+adoptive+cell+transfer+therapy.">Comparison of common gamma-chain cytokines, interleukin-2, interleukin-7, and interleukin-15 for the in vitro generation of human tumor-reactive T lymphocytes for adoptive cell transfer therapy.</a> J. Immunother. 29, 284-293 (2006).</li><li>Bear, H. D., Roberts, J., Cornell, D., Tombes, M. B., Kyle, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+immunotherapy+of+cancer+with+pharmacologically+activated+lymph+node+lymphocytes:+a+pilot+clinical+trial.">Adoptive immunotherapy of cancer with pharmacologically activated lymph node lymphocytes: a pilot clinical trial.</a> Cancer Immunol Immunother. 5, 269-274 (2001).</li><li>Morales, J. K., Kmieciak, M., Graham, L., Feldmesser, M., Bear, H. D., Manjili, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+transfer+of+HER2/neu-specific+T+cells+expanded+with+alternating+gamma+chain+cytokines+mediate+tumor+regression+when+combined+with+the+depletion+of+myeloid-derived+suppressor+cells.">Adoptive transfer of HER2/neu-specific T cells expanded with alternating gamma chain cytokines mediate tumor regression when combined with the depletion of myeloid-derived suppressor cells.</a> Cancer Immunol Immunother. 58, 941-953 (2009).</li><li>Cha, E., Graham, L., Manjili, M. H., Bear, H. D., Guy, C. T., Webster, M. A., Schaller, M., Parsons, T. J., Cardiff, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-7+++IL-15+are+superior+to+IL-2+for+the+ex+vivo+expansion+of+4T1+mammary+carcinoma-specific+T+cells+with+greater+efficacy+against+tumors+in+vivo.">IL-7 + IL-15 are superior to IL-2 for the ex vivo expansion of 4T1 mammary carcinoma-specific T cells with greater efficacy against tumors in vivo.</a> Breast Cancer Res Treat. 89, 10578-10582 (2009).</li><li>Kmieciak, M., Morales, J. K., Morales, J., Bolesta, E., Grimes, M., Manjili, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Danger+signals+and+nonself+entity+of+tumor+antigen+are+both+required+for+eliciting+effective+immune+responses+against+HER-2/neu+positive+mammary+carcinoma:+implications+for+vaccine+design.">Danger signals and nonself entity of tumor antigen are both required for eliciting effective immune responses against HER-2/neu positive mammary carcinoma: implications for vaccine design.</a> Cancer Immunol Immunother. 57, 1391-1398 (2008).</li><li>Stern, J. B., Smith, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-2+induction+of+T-cell+G1+progression+and+c-myb+expression.">Interleukin-2 induction of T-cell G1 progression and c-myb expression.</a> Science. 233, 203-206 (1986).</li><li>Kittipatarin, C., Khaled, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ex+vivo+expansion+of+memory+CD8+T+cells+from+lymph+nodes+or+spleen+through+in+vitro+culture+with+interleukin-7.">ex vivo expansion of memory CD8 T cells from lymph nodes or spleen through in vitro culture with interleukin-7.</a> J Immunol Methods. 344, 45-57 (2009).</li><li>Kokaji, A. I., Hockley, D. L., Kane, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-15+transpresentation+augments+CD8++T+cell+activation+and+is+required+for+optimal+recall+responses+by+central+memory+CD8++T+cells.">IL-15 transpresentation augments CD8+ T cell activation and is required for optimal recall responses by central memory CD8+ T cells.</a> J Immunol. 180, 4391-4401 (2008).</li><li>Le, H. K., Graham, L., Miller, C. H., Kmieciak, M., Manjili, M. H., Bear, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incubation+of+antigen-sensitized+T+lymphocytes+activated+with+bryostatin+1+++ionomycin+in+IL-7+++IL-15+increases+yield+of+cells+capable+of+inducing+regression+of+melanoma+metastases+compared+to+culture+in+IL-2.">Incubation of antigen-sensitized T lymphocytes activated with bryostatin 1 + ionomycin in IL-7 + IL-15 increases yield of cells capable of inducing regression of melanoma metastases compared to culture in IL-2.</a> Cancer Immunol Immunother. 58, 1565-1576 (2009).</li></ol>

利害の衝突は宣言されません。

エフェクターの抗腫瘍機能を持つ選択的腫瘍反応性の拡張性T細胞は、B / Iの活性化と生体外での拡張ガンマ鎖のサイトカインとIL - 2、IL - 7およびIL - 15を使用して提案されたプロトコルによって達成することができます。 IL - 2は、抗原特異的T細胞の分化と拡張をサポートすることができるT細胞増殖因子である一方、IL - 7はT細胞のアポトーシスを阻害し、拡張中にその生存をサポート?...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Bryostatin 1</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B7431-10ug</td>
<td>&nbsp;</td>
<tr>
<td>Ionomycin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>407950</td>
<td>&nbsp;</td>
<tr>
<td>Mouse IL-7</td>
<td>&nbsp;</td>
<td>PeproTech</td>
<td>217-17</td>
<td>&nbsp;</td>
<tr>
<td>Mouse IL-15</td>
<td>&nbsp;</td>
<td>PeproTech</td>
<td>210-15</td>
<td>&nbsp;</td>
<tr>
<td>Human IL-2</td>
<td>&nbsp;</td>
<td>PeproTech</td>
<td>200-02</td>
<td>&nbsp;</td>
<tr>
<td>RPMI1640</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11875</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Gemini</td>
<td>100-106</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-002-CI</td>
<td>&nbsp;</td>
<tr>
<td>L- glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030081</td>
<td>&nbsp;</td>
<tr>
<td>&beta;- mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M7522</td>
<td>&nbsp;</td>
<tr>
<td>anti-CD16/32 antibody</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>101302</td>
<td>&nbsp;</td>
<tr>
<td>Annexin V-FITC Apoptosis Detection Kit</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>556547</td>
<td>&nbsp;</td>
<tr>
<td>FITC-CD4</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>100406</td>
<td>&nbsp;</td>
<tr>
<td>PE-CD8</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>100708</td>
<td>&nbsp;</td>
<tr>
<td>anti-c-Erb2/c&#x2013;Neu</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>OP16</td>
<td>&nbsp;</td>
<tr>
<td>PE- anti mouse IgG</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>405307</td>
<td>&nbsp;</td>
<tr>
<td>formaldehyde</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>04018</td>
<td>&nbsp;</td>
<tr>
<td>Hemocytometer</td>
<td>&nbsp;</td>
<td>Hycor</td>
<td>87144</td>
<td>&nbsp;</td>
<tr>
<td>Light microscope</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>V200073</td>
<td>&nbsp;</td>
<tr>
<td>Mouse IFN-&gamma; ELISA set</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>555138</td>
<td>&nbsp;</td>
<tr>
<td>Cell culture flasks</td>
<td>&nbsp;</td>
<td>Greiner</td>
<td>658175</td>
<td>&nbsp;</td>		</tbody>
		</table>
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ex vivo expansion of tumor-reactive t cells by means of bryostatin 1/ionomycin and the common gamma chain cytokines formulation

それは、乳がんの患者は腫瘍の1,2に対する既存の免疫応答を有することが報告されました。しかし、そのような免疫応答は、乳がんの開発または再発に対する完全な保護を提供するために失敗する。腫瘍反応性T細胞の頻度を増やすことによってこの問題を克服するために、養子免疫療法が採用されている。さまざまなプロトコルは、腫瘍特異的T細胞の拡大のために使用されている。これらのプロトコルは、しかしながら、抗原特異的T細胞の活性化のための腫瘍抗原のex vivoでの使用に制限されています。ごく最近、このようなIL - 2、IL - 7、IL - 15、およびIL - 21などの一般的なγ鎖のサイトカインは、抗腫瘍免疫応答3の強化のために単独でまたは組み合わせて使用されている。しかし、製剤は腫瘍反応性T細胞の拡大のために最もよく働くなるかは明らかではない。ここでは、選択的活性化と乳がんの養子T細胞療法で使用するためにHER-2/neu陽性乳癌癌のFVBN202トランスジェニックマウスモデルからの腫瘍反応性T細胞の拡大のためのプロトコルを提示する。プロトコルは、16時間の腫瘍抗原の不在でbryostatin-1/ionomycin（B / I）とIL - 2によるT細胞の活性化が含まれています。 B / Iの活性化は細胞内シグナルを模倣してそのそれぞれ4、プロテインキナーゼC活性および細胞内カルシウムを増加させることによりT細胞活性化の結果。無関係なT細胞を殺すことながら、このプロトコルは、特に腫瘍特異的T細胞を活性化。 B / I -活性化T細胞は、24時間のIL - 7およびIL - 15で培養し、IL - 2でパルスされています。 24時間後、T細胞を洗浄し、スプリット、および追加の4日間+ IL - 15 IL - 7で培養した。 ex vivoで拡大T細胞の腫瘍特異性と抗腫瘍効果が決定されます。

Watch this Scientific Journal Video about Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation at JoVE.com

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

のための効率的なプロトコル<em&gt;生体外で</em腫瘍所属リンパ節または腫瘍を持つホストの他の二次リンパ組織から腫瘍反応性T細胞の&gt;拡張が記述されています。このプロトコルは、選択的に乳がんの養子免疫療法で使用するための腫瘍特異的T細胞を拡大する。

ブリオスタチンの1/Ionomycinによる腫瘍反応性T細胞のex vivoでの拡張と共通γ鎖のサイトカインの策定

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

ex-vivo-expansion-tumor-reactive-t-cells-means-bryostatin-1ionomycin

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JoVE Journal

Immunology and Infection

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

14.6K ビュー.  Virginia Commonwealth University- Massey Cancer Center. のための効率的なプロトコル生体外で拡張が記述されています。このプロトコルは、選択的に乳がんの養子免疫療法で使用するための腫瘍特異的T細胞を拡大する。

ビデオ: Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

HLA - Igは効率のためのベースの人工抗原提示細胞生体外で</emヒトCTLの>拡大

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

免疫・感染学

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

広視野と共焦点顕微鏡によるマウスリンパ節スライスにおけるT細胞のex vivoでのイメージング

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

ゾレドロネートを使用してヒト末梢血γδT細胞の拡大

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

ナチュラルキラーT細胞の細胞（AAPC）媒介活性化と増殖を提示する人工抗原

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

の分離<em&gt;熱帯熱マラリア原虫（Plasmodium falciparum）</em磁気手段によって&gt;後期感染赤血球

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1感染CD4の生得センシングを評価<sup&gt; +</sup&gt;使用して形質細胞様樹状細胞によるT細胞<em&gt;エクスビボ</em&gt;共培養系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

アン<em&gt;エクスビボ</em急性脳スライスにおけるオリゴデンドロサイト指向T細胞攻撃の&gt;モデル

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T細胞は、樹状細胞からTransinfectionによって細菌をキャプチャ

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

単離と<em&gt;エクスビボ</emVδ1の&gt;文化<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt;γδT細胞、胸腺外αβT細胞前駆

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.