この研究は、ミシガン大学総合がんセンターのウィルとジャンヌ・コールドウェル寄付研究基金によって、そして部分的にはNIH助成金CA82529とギルソンロンゲンボー財団によって支援されました。

1. アルデフルオール染色
<ol><li>細胞サンプル調製:培養腫瘍細胞または採取したばかりの腫瘍サンプルから、腫瘍細胞の単一細胞懸濁液を調製する。細胞を数え、ALDEFLUORアッセイバッファー内の1 x 106 細胞/mlの濃度に細胞懸濁液を調整します。</li>
	<li>コントロールチューブとして4つの12 mm x 75 mmポリスチレン試験管をラベル:#1:アンステイン、#2:アルデフルオール、#3:アルデフルオール+DEAB、および#4:7AAD。</li>
	<li>これらのコントロールチューブの場合は、1mlの細胞懸濁液を#1に入れ、#4し、チューブ#2に2mlを入れる。5 μl DEAB (ALDH阻害剤) をチューブ#3に加え、氷の上に保管します。その後、チューブ#2に10μl活性化ALDEFLUOR基質を加え、混合し、すぐに1mlの混合物をチューブ#3に移します。</li>
	<li>同時に、ALDEFLUORアッセイバッファー内の1 x 106細胞/mlの細胞(サンプルチューブ)の残りの部分にも、100万個当たりの活性化ALDEFLUOR基質を2μl添加します。</li>
	<li>4つのコントロールチューブとサンプルチューブの両方を37°Cの水浴で30分間インキュベートします。</li>
	<li>インキュベーションに続いて、5 μl 7AADをコントロールチューブ#4に加え、サンプルチューブに1 μl 7AAD 1 x 106 細胞を加えます。4°Cで10分インキュベート。</li>
	<li>250 x g で 5 分間すべてのチューブを遠心分離し、上清を除去します。</li>
	<li>ALDEFLUORアッセイバッファー内の細胞を再懸濁します。</li>
	<li>フローサイトメトリーベースのソートを実行します。</li>
	<li>DEABを負の対照として処理したALDEFLUOR染色細胞と、生存率制御のためのヨウ化プロピジウム(PI)7AAD-染色細胞を用いて選別ゲートを設定する。これらのコントロールに基づいて、ALDEFLUOR+/ALDH高、D5およびSCC7細胞を区別するゲートが確立された。これらのゲートは、ALDEFLUOR+/ALDH高D5 およびSCC7細胞のために上記で調製されたすべてのサンプル細胞をソートするために使用されます。</li>
</ol>2. がん幹細胞リセートパルス樹状細胞(CSC-TPDC)ワクチンの調製
<ol><li>CO2を用いてマウス(それぞれ同種B6およびC3H)を安楽死させ、大腿骨および脛骨を単離する。</li>
	<li>室温で1分間、骨を75%エタノールに入れます。その後、HBSSを使用して骨を洗います。</li>
	<li>骨を切り落とし、21G針と10mlの注射器を使って細胞を皿に押し込みます。</li>
	<li>吸引し、単一細胞懸濁液を作るために注射器を使用して細胞を吹き飛ばす。</li>
	<li>1,500rpmで5分間遠心分離した後、上清を捨てます。その後、RBCのリシスバッファーに5mlのRBCを加え、37°Cの水浴中に1分間RBCをライシスします。</li>
	<li>細胞数をカウントし、10 ng/ml GM-CSFおよび10 ng/ml IL-4を含む完全培地(CM)中の100万細胞/mlの濃度に細胞懸濁液を調整する。</li>
	<li>細胞を培養し、3日後にCMプラスIL-4およびGM-CSFを補償する。</li>
	<li>5日目 に、未熟樹状細胞(DC)を収穫し、4.2 ml溶液Cプラス1 ml OptiPrep濃度勾配培地を含むDC分離培地を調製する。</li>
	<li>5 ml CM で細胞ペレットを懸濁し、細胞懸濁液を分離培地にゆっくり加えます。室温で2,000rpmで遠心分離機。</li>
	<li>CM と分離メディアの間の DC を収集します。細胞数をカウントし、10 ng/ml GM-CSFおよび10ng/ml IL-4を含む培地中の100万個の細胞/mlの濃度に細胞懸濁液を調整する。</li>
	<li>ALDH高 または未ソートのD5またはSCC7細胞を培地中に懸濁させて腫瘍ライセートを調製し、その後5分間°100 x gでスピンを行い、ライセートの膜部分を回収します。</li>
	<li>CSC-TPDCを調製するために、自己ALDH高細胞のライセートを用いてDCをパルスする。H-TPDCを調製するために、不均一な異種腫瘍細胞リセートを有するパルスDC。DCと腫瘍細胞のライセートの比率は同じである。</li>
</ol>3. 有効性のワクチン接種と評価
<ol><li>正常B6マウスを2,500D5 CSC-TPDCまたはD5 H-TPDC腫瘍細胞を皮下(s.c)でそれぞれワクチンを接種する。正常なC3H動物 をワクチン 接種.c 5,000 SCC7 CSC-TPDCまたはSCC7 H-TPDC腫瘍細胞をそれぞれ有する。</li>
	<li>ワクチン接種後、20日後にCO2を用いてマウスを安楽死させる異種D5腫瘍細胞を有するB6マウスに挑戦する。肺を収穫し、肺の転移を列挙する。</li>
	<li>SCC7モデルでは、DCワクチンの反対側に未分類のSCC7腫瘍細胞 s.c を有するC3Hマウスに挑戦する。腫瘍のサイズを監視します。</li>
	<li>実験の最後にCO2を用いてB6マウスとC3Hマウスを安楽死させる。最初のワクチンの後34日目に、CO2でマウスを安楽死させ、同時に無菌処置で各群の脾臓を採取する。</li>
	<li>hrIL-2 または LPS プラス抗 CD40 (FGK45) mAb 腹水を含む CM で固定化抗 CD3 プラス抗 CD28 mAbs を使用して脾臓 T および/または B 細胞を活性化します。</li>
	<li>活性化後、上清を収集し、ELISAアッセイに使用してください。</li>
	<li>ELISAアッセイの場合、IFNγ、GM-CSFおよびIgGの抗体を4°C O/Nでコートします。</li>
	<li>ブロッキングバッファーの適用後、サンプルと標準を追加し、室温でインキュベートします。</li>
	<li>プレートを洗浄し、インキュベーション用のHRP検出抗体とTMB基質を添加します。</li>
	<li>反応を停止した後、30分以内に450 nmのELISAプレートリーダーの吸光度を測定します。</li>
</ol>

<ol>
	<li>Shafee, N., Smith, C. R., 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=Cancer+stem+cells+contribute+to+cisplatin+resistance+in+Brca1/p53-mediated+mouse+mammary+tumors.">Cancer stem cells contribute to cisplatin resistance in Brca1/p53-mediated mouse mammary tumors.</a> Cancer Res. 68, 3243-3250 (2008).</li><li>Nandi, S., Ulasov, I. V., 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=Low-dose+radiation+enhances+survivin-mediated+virotherapy+against+malignant+glioma+stem+cells.">Low-dose radiation enhances survivin-mediated virotherapy against malignant glioma stem cells.</a> Cancer Res. 68, 5778-5784 (2008).</li><li>Dallas, N. A., Xia, L., 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=Chemoresistant+colorectal+cancer+cells,+the+cancer+stem+cell+phenotype,+and+increased+sensitivity+to+insulin-like+growth+factor-I+receptor+inhibition.">Chemoresistant colorectal cancer cells, the cancer stem cell phenotype, and increased sensitivity to insulin-like growth factor-I receptor inhibition.</a> Cancer Res. 69, 1951-1957 (2009).</li><li>Yamauchi, K., Yang, 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=Induction+of+cancer+metastasis+by+cyclophosphamide+pretreatment+of+host+mice:+an+opposite+effect+of+chemotherapy.">Induction of cancer metastasis by cyclophosphamide pretreatment of host mice: an opposite effect of chemotherapy.</a> Cancer Res. 68, 516-520 (2008).</li><li>Chang, A. E., Redman, B. G., 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=A+phase+I+trial+of+tumor+lysate-pulsed+dendritic+cells+in+the+treatment+of+advanced+cancer.">A phase I trial of tumor lysate-pulsed dendritic cells in the treatment of advanced cancer.</a> Clin. Cancer Res. 8, 1021-1032 (2002).</li><li>Ito, F., Li, Q., Shreiner, A. B., 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=Anti-CD137+monoclonal+antibody+administration+augments+the+antitumor+efficacy+of+dendritic+cell-based+vaccines.">Anti-CD137 monoclonal antibody administration augments the antitumor efficacy of dendritic cell-based vaccines.</a> Cancer Res. 64, 8411-8419 (2004).</li><li>Kirk, C. J., Hartigan-O'Connor, 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=T+cell-dependent+antitumor+immunity+mediated+by+secondary+lymphoid+tissue+chemokine:+augmentation+of+dendritic+cell-based+immunotherapy.">T cell-dependent antitumor immunity mediated by secondary lymphoid tissue chemokine: augmentation of dendritic cell-based immunotherapy.</a> Cancer Res. 61, 2062-2070 (2001).</li><li>Ning, N., Pan, Q., 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=Cancer+stem+cell+vaccination+confers+significant+antitumor+immunity.">Cancer stem cell vaccination confers significant antitumor immunity.</a> Cancer Res. 72, 1853-1864 (2012).</li><li>Ginestier, C., Liu, 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=CXCR1+blockade+selectively+targets+human+breast+cancer+stem+cells+in+vitro+and+in+xenografts.">CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts.</a> J. Clin. Invest. 120, 485-497 (2010).</li><li>Carpentino, J. E., Hynes, M. J., 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=Aldehyde+dehydrogenase-expressing+colon+stem+cells+contribute+to+tumorigenesis+in+the+transition+from+colitis+to+cancer.">Aldehyde dehydrogenase-expressing colon stem cells contribute to tumorigenesis in the transition from colitis to cancer.</a> Cancer Res. 69, 8208-8215 (2009).</li><li>Charafe-Jauffret, E., Ginestier, 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=Breast+cancer+cell+lines+contain+functional+cancer+stem+cells+with+metastatic+capacity+and+a+distinct+molecular+signature.">Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature.</a> Cancer Res. 69, 1302-1313 (2009).</li></ol>

STEMCELLテクノロジーズは、オープンアクセスの出版料をサポートしています。

SCIDマウスなどの免疫不全宿主は、宿主内の適応免疫の欠如によるCSCの免疫学的評価を妨げる。本研究では、免疫能力を有する宿主におけるCSCの免疫原性を評価し、患者の設定をより厳密に模倣する可能性がある。濃縮CSCsは免疫原性であり、選択されていない腫瘍細胞のライセートパルスDCと比較して、そのライセートがワクチンとしてDCにロードされると、より効果的な腫瘍保護免疫を誘導する可能性があります。 機械学的に、CSC反応性抗体およびT細胞8 の選択的誘導ならびに1型サイトカインの産生、 例えば IFNγおよびGM-CSFの産生によって保護が与えられた。
樹状細胞ベースワクチンや養子T細胞移動を含む現在の免疫療法のほとんどは、腫瘍分化抗原を標的にするように設計されています。CSCは、これらの分化された抗原を発現しない可能性があるため、これらの免疫学的標的を免れてもよい。対照的に、がん幹細胞を特異的に標的化するように設計されたCSCワクチンは、この特殊な癌細胞集団を破壊し、腫瘍の再発および転移を予防することによってワクチンの治療効果を向上させる可能性がある。
培養した腫瘍細胞と採取したばかりの腫瘍の両方で、高アルデヒドデヒドロゲナーゼ活性に基づいて、フローサイトメトリーによりCSC濃縮集団を同定した。このようなALDH高細胞は、CSC-TPDCsを生成するためにDCをパルスする抗原源として使用されるフロー選別によって単離することができた。 これらの結果は、培養腫瘍細胞から、または臨床応用のために採取したばかりの腫瘍から分離されたCSCを使用する可能性を明らかにした。
臨床的に関連するためには、ワクチンを治療環境で調べる必要があります。これらの実験は現在、当研究室で行われています。

癌幹細胞は、従来の化学療法および放射線療法1,2に対して比較的耐性である。一方、この細胞集団は、従来の癌治療1-4後の癌の再発および進行を担う細胞である可能性がある。がん幹細胞上の分化腫瘍抗原の発現が不足しているため、がん幹細胞は、主に分化した腫瘍細胞上の抗原を標的にするように設計された癌に対する治療の現在の免疫学的介入を免れる可能性がある。したがって、がん幹細胞を特異的に標的化および破壊する新戦略の開発は、現在の癌治療の治療効果を高めるという約束を保持し得る。このため、2つの動物腫瘍(黒色腫D5および扁平上皮癌SCC7)から癌幹細胞(CSC)を豊富に含む集団を単離し、抗原提示細胞(樹状細胞、DC)をパルスする抗原源として使用し、CSC-TPDCワクチンを調製した。次に、同種免疫担当宿主、B6マウスおよびC3HマウスにおけるCSC-TPDCワクチンによって誘導された抗腫瘍免疫をそれぞれ評価した。CSC-TPDC誘導抗腫瘍効果は、我々のグループ5,6によって以前に使用された未並種の異種腫瘍細胞(H-TPDC)および他の研究者7 が前臨床試験および臨床試験の両方で使用したリセートでパルスされた従来のDCワクチンと比較した。

<table border="0" cellpadding="0" cellspacing="0" width="1022">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">ALDHEFLOUR KIT</td>
			<td style="width:399px;">Stemcell Technologies</td>
			<td style="width:219px;">1700</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Murine IL-4</td>
			<td style="width:399px;">Pepro Tech</td>
			<td style="width:219px;">214-14</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Murine GM-CSF</td>
			<td style="width:399px;">Pepro Tech</td>
			<td style="width:219px;">315-03</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Mouse IFN-&#611; ELISA KIT</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555138</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Mouse GM-CSF ELISA KIT</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555167</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">OptiPrep Density Gradient Medium</td>
			<td style="width:399px;">Sigma Aldrich</td>
			<td style="width:219px;">D1556</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">BD OptEIA TMB Substrated Reagent Set</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555214</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;"></td>
			<td style="width:399px;"></td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Equipment</td>
			<td style="width:399px;"></td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">BD FACS Aria Cell Sorter</td>
			<td>BD Biosciences</td>
			<td>336834</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Kcjunior</td>
			<td style="width:399px;">Bio-Tek Instruments</td>
			<td style="width:219px;">176058</td>
		</tr>
	</tbody>
</table>

generation of a novel dendritic-cell vaccine using melanoma and squamous cancer stem cells

我々は、マウスメラノーマD5シンジニアスからC57BL/6マウスまでの癌幹細胞(CSC)エンリッチ集団と、ALDEFLUOR/ALDHをマーカーとして使用するC3Hマウスに対する扁平上癌SCC7シンジニアスを同定し、細胞リセートをパルス樹状細胞(DC)に対する抗原の供給源として免疫原性を試験した。ALDH高CSC ライセートでパルスされたDCは、モデルと肺転移の設定およびs.cの両方で、並べ替えられていない腫瘍細胞ライセートのライセートでパルスされたDCよりも有意に高い保護抗腫瘍免疫を誘導 した。腫瘍の成長設定, それぞれ.この現象は、CSCワクチン誘発性体液性ならびに細胞抗CSC応答によるものであった。特に、CSC-DCワクチンを受けた宿主から単離された脾細胞は、パルスドDCワクチンを受けた宿主から単離された脾細胞よりも有意に高い量のIFNγおよびGM-CSFを産生した。これらの結果は、アジュバントの設定で臨床使用のための自家CSCベースの治療ワクチンを開発するための努力を支持する。

ALDEFLUOR/ALDHは、多発性悪性腫瘍8-11で幹細胞を単一マーカーとして使用されている。我々は、ALDEFLUORをマーカーとして用い、2つの腫瘍モデルD5およびSCC7における癌幹細胞濃縮集団を同定した。マウス黒色腫B16-D5および扁平上皮癌SCC7のALDEFLUOR+細胞を検出した。ALDEFLUOR+細胞が、それぞれ培養されたD5およびSCC7の腫瘍細胞株に約0.5%および5.2%の寄与を示した(図1)。確立された腫瘍から採取したばかりの腫瘍細胞を分析し、ALDEFLUOR+細胞の存在を確認した。 図1にも示すように、 インビボ 樹立D5およびSCC7腫瘍からALDEFLUOR+細胞の2.5%および4.2%がそれぞれ存在した。これらの分類D5およびSCC7 ALDEFLUOR+/ALDHの高い 集団の腫瘍原性および自己再生能力を、同系免疫担当宿主、C57BL/6およびC3Hマウスでそれぞれ8個評価した。
我々は、動物実験と臨床試験5,6の両方で、異種の未分類の腫瘍細胞ライセートをパルスDC(H-TPDC)に使用した。CSCの免疫原性を調べるために、ALDH高 CSCを単離し、CSCのリセートでDCをパルスしてCSC-TPDCを生成し(図2)、従来のがんワクチンコントロールとしてH-TPDCを使用して、CSC-TPDCが腫瘍増殖を予防するのに有益な効果があるかどうかを検査しました。
CSC-TPDCによる保護抗腫瘍免疫を調べることにより、CSCの免疫原性を評価した。D5モデルでは、ナイーブ免疫担当マウスをs.c CSC-TPDCまたはH-TPDC(同じlysate:DC比)でワクチン接種した。 制御グループは生理的な(PBS)を受け取った。最後のワクチンの1週間後、マウスは未分類のD5腫瘍細胞を静脈内(すなわち)で挑戦し、肺を3週間後に採取して肺転移を列挙した。表1に示すように、PBS群と比較して、H-TPDCで治療されたマウスは肺転移が少ない。重要なことに、CSC-TPDCで治療されたマウスは、両方の実験でH-TPDCワクチン群よりも肺転移が有意に少なかった。SCC7モデルでは、正常なC3H動物をs.cにSCC7 CSC-TPDCまたはH-TPDCをそれぞれ右脇腹にワクチン接種し、続いて未ソートのSCC7腫瘍細胞を左脇腹に.cに挑戦した。PBS群と比較して、H-TPDCは腫瘍増殖に対して控えめな抗腫瘍免疫を誘導した。しかし、コントロール群とH-TPDC群の両方と比較した場合にCSC-TPDCで治療したマウスでは腫瘍増殖の有意な抑制があった(p<0.05,図3)。これらの結果は、CSCが腫瘍細胞の挑戦に対する保護免疫を誘導する従来の未分類の腫瘍細胞よりもDCをロードするより効果的な抗原源として使用できることを示している。
観察されたCSC誘導保護抗腫瘍免疫の根底にあるメカニズムをさらに理解するために、実験の最後にDCワクチン接種を受けた動物から脾臓を採取した。脾臓細胞は、抗CD3/CD28/IL-2または抗CD3/CD28/IL-2+ LPS/抗CD40によって活性化された。次いで、培養上清を収集し、サイトカインおよび抗体の発現を検出した。 D5 CSC-TPDCまたはSCC7 CSC-TPDCでワクチン接種された動物から脾臓細胞によるIFNγおよびGM-CSFの産生が有意に高かった(図4)。さらに、D5 H-TPDCまたはSCC7 H-TPDCと比較して、D5 CSC-TPDCまたはSCC7 CSC-TPDCでワクチン接種された動物から採取されたLPS/抗CD40活性化脾臓細胞によるIgG産生が有意に(p<0.05)高かった。これらの抗体は、それぞれD5およびSCC7 CSCに結合することが判明し、そのような結合は、補体8の存在下でCSCリシスをもたらす可能性がある。
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig1highres.jpg" src="/files/ftp_upload/50561/50561fig1.jpg"> 図 1. ALDHFluOR+/ALDH高集団 は、培養された新鮮なマウスD5黒色腫およびSCC7扁平上皮細胞腫瘍と同様に、高い集団を検出した。 腫瘍細胞は、特定のALDH阻害剤である50 mmol/L DEABで治療し、陰性対照として使用した。
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig1highres.jpg" target="_blank">ここをクリックすると、より大きな画像が表示されます。</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig2highres.jpg" src="/files/ftp_upload/50561/50561fig2.jpg"> 図 2. 樹状細胞系癌幹細胞ワクチンの生成 CSC-TPDCおよびH-TPDCの調製のために、骨髄由来樹状細胞をそれぞれALDH高 または未分類の腫瘍細胞ライセートでパルスした。
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig2highres.jpg" target="_blank">ここをクリックすると、より大きな画像が表示されます。</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig3highres.jpg" src="/files/ftp_upload/50561/50561fig3.jpg"> 図 3. CSCリセートパルスDC(CSC-TPDC)ワクチンは 、s.c においてより効果的な保護抗腫瘍免疫を誘導する可能性がある。SCC7腫瘍モデル。
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig3highres.jpg" target="_blank">ここをクリックすると、より大きな画像が表示されます。</a>
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig4highres.jpg" src="/files/ftp_upload/50561/50561fig4.jpg"> 図 4. CSC-TPDCでワクチン接種された免疫能力宿主におけるより強力な全身細胞応答. 脾細胞は、H-TPDCまたはCSC-TPDCワクチン接種を受けた動物から採取し、示されるように活性化した。次いで、ELISAを用いてサイトカイン検出のために培養上清を回収した。
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig4highres.jpg" target="_blank">ここをクリックすると、より大きな画像が表示されます。</a>

Watch this Scientific Journal Video about Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells at JoVE.com

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

同系免疫担当宿主において評価された癌幹細胞(CSC)ベース樹状細胞(DC)ワクチンは、異種バルク腫瘍細胞でパルスされた従来のDCワクチンよりも有意に高い抗腫瘍免疫を示した。

黒色腫と扁平上癌幹細胞を用いた新規樹状細胞ワクチンの生成

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

generation-novel-dendritic-cell-vaccine-using-melanoma-squamous

Research

JoVE Journal

Immunology and Infection

11.2K ビュー.  University of Michigan. 同系免疫担当宿主において評価された癌幹細胞(CSC)ベース樹状細胞(DC)ワクチンは、異種バルク腫瘍細胞でパルスされた従来のDCワクチンよりも有意に高い抗腫瘍免疫を示した。

ビデオ: Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

アレルギー性​​気道炎症時の肺樹状細胞の成熟および移行の解析

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

免疫・感染学

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

スフィンゴ糖脂質抗原の質量分析

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

全血の表現型アッセイを用いて多施設臨床試験のための主要な末梢血白血球集団の列挙

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

使用して樹状細胞の移行の監視<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁気共鳴イメージング

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

FDAの承認を受けたVero細胞におけるワクチン開発のための組換えアレナウイルスの生成

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

精製造血幹細胞/前駆細胞のDNAの変異を特定する

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

宿主細胞のRNA合成でウイルス感染の効果を測定するためにクリックケミストリーを用いた

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

サイトカインキャプチャシステムを用いた臨床応用のためのサイトメガロウイルス特異的T細胞の自動化された細胞濃縮

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

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 ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

大量の骨髄前駆細胞と新規セルソーティング戦略を使用してマウスの骨髄から樹状細胞前駆体の生成

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...