저자는 폴리 ICLC의 선물 안드레스 살라을 (Oncovir, 주식 회사) 감사드립니다.

1. 말초 혈액 4의 분리 및 냉동 <ol><li> 무균 스파이크 플라즈마 전송 집합을 사용하여 leukapheresis 부대에있는 액세스 포트 중 하나입니다. 60 ML의 주사기를 사용하여 멸균 500 ML 병에 환자로부터 얻은 leukapheresis를 전송합니다. </li><li> 상온 RPMI를 사용하여 원래 볼륨을 2 배 leukapheresis의 볼륨을 조정합니다. 철저하게 섞는다. </li><li> 부드럽게 Ficoll-Paque PLUS의 병을 섞는다. 멸균 50 ML 원뿔 튜브에 12 ML Ficoll-Paque PLUS를 추가합니다. </li><li> Ficoll을 포함하는 각 멸균 50 ML 원뿔 튜브에 희석 leukapheresis 제품의 부드럽게 층 30 ML. Ficoll과 세포 현탁액 사이의 인터페이스를 방해하지 않도록주의하십시오. </li><li> leukapheresis의 모든 계층 될 때까지 단계 1.3 및 1.4를 반복합니다. </li><li> 없이 브레이크와 실온에서 20 분간 1,000 × g에서 50 ML 원뿔 튜브를 원심 분리기. </li><li> 조심스럽게 각 튜브 및 transfe에서 말초 혈액의 흐림 층을 수확새로운 멸균 50 ML 튜브에 대한 연구. </li><li> 50 ML의 최종 볼륨에 각 튜브에 RPMI를 추가합니다. 반전에 의해 부드럽게 섞는다. </li><li> 4 10 분 ° C 풀 브레이크를 위해 500 × g에서 세포를 원심 분리기. </li><li> 각 튜브에서 상층 액을 제거합니다. 각 튜브에서 세포 펠렛을 Resuspend하고 50 ML의 최종 볼륨에 RPMI를 추가합니다. 반전에 의해 부드럽게 섞는다. </li><li> 4시 6 분 ° C.에 대한 500 × g에서 세포를 원심 분리 </li><li> 각 튜브에서 상층 액을 제거합니다. 를 Resuspend 한 50 ML 원뿔 튜브에 함께 풀 세포 현탁액. </li><li> 위로 더 RPMI 50 ML에 풀 말초 혈액의 양을 가져옵니다. 반전에 의해 부드럽게 섞는다. </li><li> 4시 6 분 ° C.에 대한 300 × g에서 세포를 원심 분리 </li><li> 상층 액을 제거합니다. 50 ML RPMI에있는 세포 펠렛을 resuspend. 균일 한 세포 현탁액을 보장하기 위해 부드럽게 섞는다. </li><li> 세포의 수를 계산하고 세포 생존을 결정합니다. 가능한 세포의 총 수를 계산합니다. </li><li> 300에서 세포를 원심 분리4시 6 분 × g ° C. 냉동 매체를 준비합니다. 인간 AB 혈청 (밸리 생명)에서, 냉동 매체는 10 % 디메틸 설폭 사이드 (DMSO있는 Miltenyi Biotec은)으로 구성되어 있습니다. </li><li> 상층 액을 제거합니다. 2 × 10 8 세포 / 차가운 냉동 미디어 ML의 최종 농도에서 세포를 resuspend을. </li><li> 1.8 ML의 cryovials 1 ML의 aliquots을 확인합니다. </li><li> 제어 속도 냉동실에 cryovials를 전송하고 동결 실행, 프로그램 시작 1. 실행의 끝에, 즉시 액체 질소 냉동고의 기상에 냉동 cryovials를 전송합니다. </li></ol> 2. 일 0 단핵구에서 수지상 세포의 분화 <ol><li> 멸균 50 ML 원뿔 튜브에 30 RPMI의 ML / 1 %자가 혈장을 추가합니다. </li><li> 37 ° C의 물을 욕조에서 교반하여 냉동 말초 혈액의 해동 분주. 완전히 해동 할 때, 멸균 50 ML 원뿔 튜브에 튜브의 내용을 전송합니다. 철저하게 섞는다. </li><li> 6 분 동안 세포를 원심 분리상온에서 500 × g에서. 상층 액을 제거하고 RPMI / 1 %자가 혈장의 작은 볼륨 펠렛 세포를 resuspend을. 다음 50 ML의 최종 볼륨에 더 RPMI / 1 %자가 혈장을 추가합니다. 철저하게 섞는다. </li><li> 500 6 분 × g, 실온에서 다시 세포를 원심 분리기. 뜨는을 대기음 50 ML RPMI / 1 %자가 혈장 펠렛을 resuspend을. 철저하게 섞는다. </li><li> 세포의 수를 계산하고 세포 생존을 결정합니다. 가능한 세포의 총 수를 계산합니다. </li><li> 판 1.40 40 RPMI의 ML / 225cm 2 EasyFlask 위에 1 %자가 혈장에서 x 10 8 세포. 모든 세포가 도금 될 때까지 반복합니다. </li><li> 1 옆으로 납작 EasyFlasks 품다 - 37 2 시간을 ° C 조직 문화 인큐베이터는 단핵구가 술병을 준수 할 수 있도록 5 % CO 2를 포함합니다. </li><li> 배양이 완료되면, 인큐베이터에서 EasyFlask을 제거하고 비 부착 세포에게 USI 제거를 두 번 세척prewarmed RPMI의 NG 30 ML. </li><li> 두 번째 세척 후, 400-1,000 IU / ML IL-4, 100-1,000 IU / ㎖ GM-CSF 5-8 40 RPMI의 ML / 1 %자가 혈장을 추가합니다. 이 프로토콜에 대한, 우리는 400 IU / ML IL-4, 100 IU / ㎖ GM-CSF를 사용하고 있습니다. </li><li> 37 2 일간 EasyFlasks을 품어 ° 조직 문화 인큐베이터에서 C가 5 % CO 2를 포함. </li></ol> 3. 주 2 : IL-4, GM-CSF와 수지상 세포의 먹이 <ol><li> 각 EasyFlask에 400-1,000 IU / ML IL-4, 100-1,000 IU / ㎖ GM-CSF와 4 RPMI의 ML / 1 %자가 혈장을 추가합니다. 소용돌이 옆으로 흔들어 섞는다. </li><li> 37 ° C 조직 문화 인큐베이터에서 5 % CO 2를 포함에서 더보기 3 일 EasyFlasks을 (5 일까지) 품어. </li></ol> 4. 5 일 : 미성숙 수지상 세포의 수확 <ol><li> 적극적 플라스크 소용돌이에 의해 비 부착 느슨하게 자기편 세포를 resuspend을하고 위아래로 pipetting하여 문화를 수확. 를 전송새로운 50 ML 원뿔 튜브에 수확 세포. </li><li> 상온에서 6 분간 500 × g에서 튜브를 원심 분리기. 상층 액을 제거하고 50 ML RPMI / 1 %자가 혈장에있는 세포 펠렛을 resuspend. 철저하게 섞는다. </li><li> 세포의 수를 계산하고 세포 생존을 결정합니다. 가능한 세포의 총 수를 계산합니다. </li><li> 상온에서 6 분간 500 × g에서 튜브를 원심 분리기. 플레이트 1-2 × 10 400 IU / ML IL-4 및 6 - 잘 조직 배양 플레이트에 100 IU / ㎖ GM-CSF 3 ML RPMI / 1 %자가 혈장에서 잘 당 6 셀. </li><li> 37 접시를 품어 ° C는 조직 문화 인큐베이터 2 밤새 5 % CO를 포함. </li></ol> 5. 6 일 : 수지상 세포의 성숙과 항원 로딩 중 <ol><li> 10 ㎍ / ㎖ KLH에 6 자 문화 접시에있는 우물의 1 / 3 추가 할 수 있습니다. 소용돌이 판 섞어. KLH는 첫 번째 DC 백신에 사용되기 때문에 KLH는 우물의 1 / 3에 추가됩니다. 이후 DC 백신 콘만 종양 항원 펩티드를 테인. </li><li> 수지상 세포는 다양한 자극을 사용하여 성숙 자극 할 수 있습니다. 임상 연구에 사용 된 가장 일반적인 성숙 자극 크린 시토 킨의 칵테일 (TNFα, IL-1 β, 및 IL-6)과 프로스타글란딘 E2 (PGE 2) 9,10왔다. 더 최근에, 수신자 같은 수용체 (TLR) 작용제의 사용은 인기 11,12를 얻고있다. 이 프로토콜에서는, 우물 2 MG / ML Polyinosinic-Polycytidylic 산성 폴리-L-라이신 카르복시 메틸 (폴리 ICLC)를 추가하고 잘 섞는다. 폴리 ICLC 폴리-L-*-라이신 및 카르복시 메틸 셀룰로오스 (Oncovir, 주식 회사에 의해 제조)로 안정화 폴리 IC의 임상 등급 정립이다. </li><li> 각 잘 간 경쟁 13을 피하기 위해 단일 펩티드를 받고, 그 지정 우물에 100 ㎍ / ㎖ 긴 펩티드 항원 (NY-ESO-1 및 / 또는 Melan-A/MART-1)를 추가합니다. </li><li> 37 접시를 품어 조직 문화 인큐베이터는 5 % CO를 포함 10 ° C &lt;서브&gt; 2 O / N. </li></ol> 6. 7 일 : 수지상 세포의 냉동 보존 <ol><li> 자가 혈장과 10 % DMSO를 사용하여 DC 냉동 솔루션을 준비합니다. 모든 미립자 물질을 제거하기 위해 4 ° C에서 20 분 동안 2,000 DC 냉동 솔루션 × g의 원심 분리기. 새로운 라벨 15 ML 원뿔 튜브에 뜨는을 전송합니다. 4에서 보관 ° 사용까지 C. </li><li> 하룻밤 배양이 완료되면, 수확 및 수영장 50 ML 원뿔 튜브에 펩타이드와 펄스 DC가 포함 된 모든 우물. </li><li> 6 분 동안 500 × g에서 튜브를 원심 분리기. 상층 액을 제거합니다. 5 RPMI의 ML / 1 %자가 혈장에있는 세포 펠렛을 resuspend. 최대 RPMI / 1 %자가 플라즈마 50ml로 전체 볼륨을 가져와. 철저하게 섞는다. </li><li> 세포의 수를 계산하고 세포 생존을 결정합니다. 가능한 세포의 총 수를 계산합니다. </li><li> 상온에서 6 분간 500 × g에서 튜브를 원심 분리기. 상층 액을 제거하고 5 ML steri의 각 펠렛을 resuspend을르 0.9 % 염화나트륨, USP 새로운 15 ML 원뿔 튜브로 전송. 더 멸균 0.9 % 염화나트륨, USP와 함께 14 ML 각 세포 현탁액을 가져옵니다. 반전에 의한 모자 및 섞는다. </li><li> 이전 단계를 두 번 반복합니다. 마지막으로 원심 분리 한 후 펠렛을 방해하지 않고 최대한 가까이 세포 펠렛을 받고, 상층 액을 제거하십시오. </li><li> 4시에 DC 냉동 용액에 세포 펠렛을 Resuspend - 20 X 10 6 세포 / 각 1.8 ML의 CryoTube 유리 병에 ML 및 나누어지는 1 ML. </li><li> DC 백신의 분주을 동결하고 장기 저장을위한 액체 질소 냉동고의 기상으로 즉시 전송, 제어 속도 냉장고, 프로그램 사용 1. </li></ol> 7. 많은 릴리스 테스트 <ol><li> DC 백신의 각 배치를 평가하고 일반적으로 5~6주 백신 준비 후 연구에서 환자에 주입 해제되기 전에 특정 로트 출하 기준 (표 1)을 만족해야합니다. </li><li> 생존 :자동 셀 카운터를 사용하여 DC 백신의 가능성을 평가한다. 최소 허용 가능성 사양&gt; 70 %이다. </li><li> 식별 : 유동 세포 계측법에 의해 성숙 수지상 세포 (CD11c + CD14-CD83 +)의 신원을 평가합니다. CD11c의 + 세포의 비율&gt; 75 %이어야한다. CD11c + 및 CD14 + 세포의 비율이되어야합니다 &lt;30 % CD11c의 비율 +, CD14-, 및 CD83 + 세포가 있어야한다&gt; 75 %. </li><li> 불임 : 직접 문화 그램 얼룩에 의한 혐기성과 호기성 세균과 곰팡이의 존재에 대한 테스트 DC 백신. 직접 배양 시스템 및 지표 세포주 DNA 형광 염색 분석을 사용하여 마이코 플라스마의 존재를 평가한다. 이러한 테스트의 최소 사양 집합은 성장이 모든 문화에서 관찰되는 것입니다. </li><li> 독소 : 운동-QCL 운동 발색 LAL 분석을 사용하여 독소의 내용을 평가하고 최소한의 기준을 충족 &lt;50 EU / ML해야합니다. </li><lI&gt; 기능 : 동종 T 세포와 함께 배양하여 혼합 림프구 반응 DC 기능을 평가합니다. 자극받지 않은 수지상 세포에 비해 증식의 존재는 (그림 3)보고됩니다. </li></ol>

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Immunol. 15, 120-127 (2003).</li><li>Bogunovic, 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=TLR4+engagement+during+TLR3-induced+proinflammatory+signaling+in+dendritic+cells+promotes+IL-10-mediated+suppression+of+antitumor+immunity.">TLR4 engagement during TLR3-induced proinflammatory signaling in dendritic cells promotes IL-10-mediated suppression of antitumor immunity.</a> Cancer Research. 71, 5467-5476 (2011).</li><li>Verdijk, R. 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=Polyriboinosinic+polyribocytidylic+acid+(poly(I:C))+induces+stable+maturation+of+functionally+active+human+dendritic+cells.">Polyriboinosinic polyribocytidylic acid (poly(I:C)) induces stable maturation of functionally active human dendritic cells.</a> J. Immunol. 163, 57-61 (1999).</li><li>Rouas, 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=Poly(I:C)+used+for+human+dendritic+cell+maturation+preserves+their+ability+to+secondarily+secrete+bioactive+IL-12.">Poly(I:C) used for human dendritic cell maturation preserves their ability to secondarily secrete bioactive IL-12.</a> Int. Immunol. 16, 767-773 (2004).</li><li>Jongmans, W., Tiemessen, D. M., van Vlodrop, I. J., Mulders, P. F., Oosterwijk, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th1-polarizing+capacity+of+clinical-grade+dendritic+cells+is+triggered+by+Ribomunyl+but+is+compromised+by+PGE2:+the+importance+of+maturation+cocktails.">Th1-polarizing capacity of clinical-grade dendritic cells is triggered by Ribomunyl but is compromised by PGE2: the importance of maturation cocktails.</a> J. Immunother. 28, 480-487 (2005).</li><li>Krause, P., 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=Prostaglandin+E2+is+a+key+factor+for+monocyte-derived+dendritic+cell+maturation:+enhanced+T+cell+stimulatory+capacity+despite+IDO.">Prostaglandin E2 is a key factor for monocyte-derived dendritic cell maturation: enhanced T cell stimulatory capacity despite IDO.</a> J. Leukoc. Biol. 82, 1106-1114 (2007).</li><li>Morelli, A. E., Thomson, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+under+the+spell+of+prostaglandins.">Dendritic cells under the spell of prostaglandins.</a> Trends Immunol. 24, 108-111 (2003).</li><li>Adams, 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=Dendritic+cell+(DC)+based+therapy+for+cervical+cancer:+use+of+DC+pulsed+with+tumour+lysate+and+matured+with+a+novel+synthetic+clinically+non-toxic+double+stranded+RNA+analogue+poly+[I]:poly+[C(12)U]+(Ampligen+R).">Dendritic cell (DC) based therapy for cervical cancer: use of DC pulsed with tumour lysate and matured with a novel synthetic clinically non-toxic double stranded RNA analogue poly [I]:poly [C(12)U] (Ampligen R).</a> Vaccine. 21 (12), 787-790 (2003).</li><li>Colombo, M. P., Trinchieri, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-12+in+anti-tumor+immunity+and+immunotherapy.">Interleukin-12 in anti-tumor immunity and immunotherapy.</a> Cytokine Growth Factor Rev. 13, 155-168 (2002).</li><li>Mayordomo, J. I., 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=Bone+marrow-derived+dendritic+cells+pulsed+with+synthetic+tumour+peptides+elicit+protective+and+therapeutic+antitumour+immunity.">Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity.</a> Nat Med. 1, 1297-1302 (1995).</li><li>Dhodapkar, M. 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=Rapid+generation+of+broad+T-cell+immunity+in+humans+after+a+single+injection+of+mature+dendritic+cells.">Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells.</a> J. Clin. Invest. 104, 173-180 (1999).</li><li>Schuler-Thurner, 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=Rapid+induction+of+tumor-specific+type+1+T+helper+cells+in+metastatic+melanoma+patients+by+vaccination+with+mature,+cryopreserved,+peptide-loaded+monocyte-derived+dendritic+cells.">Rapid induction of tumor-specific type 1 T helper cells in metastatic melanoma patients by vaccination with mature, cryopreserved, peptide-loaded monocyte-derived dendritic cells.</a> J. Exp. Med. 195, 1279-1288 (2002).</li></ol>

저자는 다음에서 재정 지원을받은 : NIH (AI061684, AI071078, AI044628, 및 K08 AI084578 (밀러)), 빌과 멜린다 게이츠 재단, 도리스 듀크 자선 재단, 암 연구소, 루퍼스 연구를위한 동맹, 그리고 에메랄드 재단. 니나 Bhardwaj는 자료 및 내성을 조작하는 수지상 세포의 사용과 관련된 특허에 coinventor입니다. 저자는 다른 관련 제휴 나 금융 관심이나 떨어져 개시된에서 원고에서 논의 된 주제 나 자료와 재무 충돌 어떤 조직이나 기업과 금융 참여가 없습니다.

단계 I 및 II는 단핵구 유래 수지상 세포의 임상 시험은 환자 그러나 임상 적 성공은 1 제한되었습니다에 면역 반응을 유도하는 것으로 나타났습니다. 이 종양 immunotherapeutic 사용을위한 최적의 수지상 세포를 생성하는 방법에 대한 합의의 부족으로 인해 부분적으로 할 수 있습니다. 임상 등급 수지상 세포를 생성하는 여러 가지 방법이 있지만,이 방법은 단핵 세포 성숙을 유도하는 데 자극 ...

<table border="1">
<tbody>
 <tr>
 <td fo:width="2in">Reagent/Supplies/Equipment</td>
 <td>Manufacturer</td>
 <td>Catalog No.</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI-1640 medium with L-glutamine</td>
 <td>BioWhittaker</td>
 <td>12-702F</td>
 <td></td>
 </tr>
 <tr>
 <td>1M HEPES buffered saline</td>
 <td>BioWhittaker</td>
 <td>17-737E</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate buffered saline (PBS)</td>
 <td>BioWhittaker</td>
 <td>17-516F</td>
 <td></td>
 </tr>
 <tr>
 <td>Human albumin, 25% solution USP</td>
 <td>Aventis Behring</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ficoll-Hypaque PREMIUM</td>
 <td>GE Healthcare</td>
 <td>17-5442-03</td>
 <td></td>
 </tr>
 <tr>
 <td>Human AB serum</td>
 <td>Valley Biomedical</td>
 <td>HP1022</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile saline USP</td>
 <td>Hospira</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>CryoMACS DMSO </td>
 <td>Miltenyi Biotec</td>
 <td>170-076-303</td>
 <td></td>
 </tr>
 <tr>
 <td>Leukine GM-CSF, 0.5 mg/ml</td>
 <td>Berlex</td>
 <td>A02266</td>
 <td></td>
 </tr>
 <tr>
 <td>MACS GMP IL-4</td>
 <td>Miltenyi Biotec</td>
 <td>170-076-101</td>
 <td></td>
 </tr>
 <tr>
 <td>Hiltonol, Poly-ICLC, 2 mg/ml</td>
 <td>Oncovir</td>
 <td>NA</td>
 <td></td>
 </tr>
 <tr>
 <td>VACMUNE KLH</td>
 <td>Biosyn</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>225 sq cm EasyFlasks</td>
 <td>Nalgene Nunc</td>
 <td>159934</td>
 <td></td>
 </tr>
 <tr>
 <td>Falcon 6-well tissue culture plates</td>
 <td>Becton Dickinson</td>
 <td>353046</td>
 <td></td>
 </tr>
 <tr>
 <td>1.8 ml CryoTube vials</td>
 <td>Nalgene Nunc</td>
 <td>377267</td>
 <td></td>
 </tr>
 <tr>
 <td>Controlled Rate Freezer</td>
 <td>Thermo</td>
 <td>CryoMed</td>
 <td></td>
 </tr>
 </tbody>
</table>

preparation of tumor antigen-loaded mature dendritic cells for immunotherapy

임상 연구는 항원 -로드 DC 백신은 안전하고 종양 1 유망한 치료 것을 설치하는 동안, 그들의 임상 효능은 확립되어야 남아있다. 이 방법은, 좋은 제조 공정 (GMP) 지침에 따라 준비, 아래에 설명 된 임상 연구 2 수지상 세포의 큰 숫자를 생성하기위한 가장 일반적인 전직 생체 제조 방법의 최적화입니다. 우리의 방법은 합성 TLR 3 작용제 수지상 세포를 자극하는 Polyinosinic-Polycytidylic 산성 폴리-L-라이신 카복시 메틸 셀룰로오스를 (폴리 ICLC)를 사용합니다. 우리의 이전 연구는 CD83과 CD86, 인터루킨-12의 유도 (IL-12), 종양 괴사 인자 (TNF), 인터페론 감마 유도의 상향 조절에 의해 평가로 폴리 ICLC 인간 DC에 대한 가장 강력한 개인의 성숙 자극이다 설립 단백질 10 (IP-10), interleukmin 1 (IL-1) 및 유형 I 인터페론 (IFN), 그리고 최소한의 인터루킨 10 (IL-10) 생산. </p&gt; 수지상 세포는 채집 술에 의해 얻어진 림프구가 냉동 말초 혈액 단핵 세포 (말초 혈액)에서 차별화된다. 말초 혈액을 Ficoll 기울기 원심 분리하여 분리 및 분취에 고정되어 있습니다. 1 일에서 말초 혈액을 해동하고 ° C에서 조직 문화 인큐베이터 37 1-2 시간 배양 한 후 플라스틱 표면에 부착 단핵구를 위해 선택하는 조직 문화 플라스크에 도금. 배양 후 림프구는 씻어하고 부착 단핵 세포는 인터루킨-4의 존재 (IL-4)와 미성숙 수지상 세포로 분화하는 과립구 대 식세포 콜로니 자극 인자 (GM-CSF) 5 일 동안 배양한다. 6 일에, 미성숙 수지상 세포는 백신의 품질에 대한 관리 역할 및 백신 3의 면역 원성을 높일 수있는 열쇠 구멍 삿갓 조개 헤 모시 아닌 (KLH) 단백질과 펄스 있습니다. 수지상 세포는 성숙 자극 펩티드 항원로드 하룻밤 배양한다. 7 일에 세포를 세척하고 포함 1 ML의 분주에 냉동4 - 제어 속도 냉동고를 사용하여 20 X 10 6 세포. 수지상 세포의 배치를위한 방출 시험이 수행되고 그들이 환자에 주입되기 전에 최소 사양을 충족해야합니다.

10 사이 - 시작 말초 혈액의 20 %는 문화 시대의 끝에서 수지상 세포로 분화. 성숙 수지상 세포는 CD11c 있습니다 +, CD14-, CD83 +, CD40 +, 및 CCR7 + (그림 1). 그들은 MHC 클래스 I 및 II 분자와 보조 자극 분자 CD80 및 CD86의 높은 수준을 표현한다. 다른 TLR 길항제 (14)에 비해 폴리 ICLC는 PDL-1의 낮은 수준을 유도. 또한 이러한 폴리 IC-성숙 수지상 세포 분비 큰 IL-12의 양의 (그림 2, ...

Watch this Scientific Journal Video about Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy at JoVE.com

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

종양 면역 요법에 사용하기 위해자가 수지상 세포의 큰 숫자 (DCS)를 생성하기위한 가장 일반적으로 사용되는 방법을 설명합니다. 방법은 단핵 세포에서 수지상 세포를 분화하는 IL-4, GM-CSF를 사용합니다. 그들은 환자에게 다시 주입하기 전에 미성숙 수지상 세포는 성숙하고 항원과 함께로드를 자극한다.

면역 요법에 대한 종양 항원로드 성숙 수지상 세포의 준비

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

preparation-tumor-antigen-loaded-mature-dendritic-cells-for

Research

JoVE Journal

Immunology and Infection

18.1K 조회수.  NYU Langone Medical Center. 종양 면역 요법에 사용하기 위해자가 수지상 세포의 큰 숫자 (DCS)를 생성하기위한 가장 일반적으로 사용되는 방법을 설명합니다. 방법은 단핵 세포에서 수지상 세포를 분화하는 IL-4, GM-CSF를 사용합니다. 그들은 환자에게 다시 주입하기 전에 미성숙 수지상 세포는 성숙하고 항원과 함께로드를 자극한다.

비디오: Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

B 세포의 CD40 - 정품 인증 Murine 모델

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

연구

면역학 및 감염병학

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

세포 성숙없이 돌기 세포의 효율적인 Transfection을 위해 최적화된 프로토콜

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

마우스 폐에 돌기 세포의 분리

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

인간의 흉선에서 골수 수지상 세포와 상피 세포의 분리

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

배려의 아교 모세포종 표준의 맥락에서 입양 치료에 대한 자동차 T 세포의 생성

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

대사 표현형을 각기 다른 미성숙, 성숙 및 Tolerogenic 수지상 세포의 생성

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

와 감염시 배수 림프절에 쥐 피부 수지상 세포의 마이그레이션을 연구하는 CFSE 기반 분석<em&gt; 소 결핵균</em&gt; Bacille 칼 메트 - GUERIN

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

전혈로부터 인간 단핵구 유래 수지상 세포의 생성

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

개발 및 검증 磁 단일 분자의 배열 디지털 효소 연결 된 Immunosorbent 분석 결과 인간 인터페론-α에 대 한

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

생리 적 면역 원 성 수지상 세포 생성을 위한 새로운 프로토콜

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...