1. PBMC의 분리 <ol><li> BD Vacutainer CPT 셀 준비 튜브 나트륨 헤파린과에 혈액 (7.5-8.0 ML)를 그립니다. 튜브는 나트륨 헤파린의 항응고제와 Ficoll - Hypaque 밀도 유체, 더하기 두 액체를 분리 폴리에스터 겔 장벽을 포함하고 있습니다. 1,800 X g에서 20 분 수평 로터 (스윙 아웃 헤드)에 실온 (18 ° C 25 ° C)에서 튜브 / 혈액 샘플을 원심 분리기. 브레이크를 원심 분리기 전환합니다. </li><li> 원심 분리 후, 레이어의 순서 (위에서 아래에서 본) 다음과 같이 발생합니다 :) 플라즈마 - B) 말초 혈액 mononuclear 세포 (PBMC)과 혈소판 - C) 밀도 솔루션 - D) 폴리에스터 겔 - E) granulocytes - F) 적혈구 (그림 1). </li><li> 셀 계층을 방해하지 않고 계면 위의 플라즈마의 5~10mm를 떠나, 플라즈마 층의 일부를 수집합니다. 플라즈마는 문화 (섹션 2.5)을 위해 사용될 수 있습니다. </li><li> 15 ML 원뿔 관에 피펫 및 이전과 계면에서 농축 분율 (PBMC)를 수확. </li><li> 10 ML과 PBMC을 씻 는다 인산염 버퍼 반전 튜브에 의해 생리 (PBS), 5 번, 그리고 400 X g에서 5 분 원심 분리기. </li><li> 두 번 세척 단계를 반복한 다음 PBS 5 ML에있는 세포 펠렛을 resuspend. 휴대폰 번호를 확인합니다. 보통 1.3 X10 6 셀은 전체 혈액의 1 ML에서 회복됩니다. </li><li> 주파수 및 유동세포계측법 (섹션 3) (그림 2)에 의해 PBMC에서 γδ T 세포의 표현형를 결정합니다. </li></ol> 2. γδ ​​T 세포의 확장 <ol><li> 상온에서 400 X g에서 5 분 15 ML 원뿔 튜브에 세포를 원심 분리기 suspensions하고 supernatants 폐기하십시오. </li><li> 각각 1000 IU / ML 5 μm의 최종 농도에 인간 IL - 2 (IL - 2)와 zoledronate (Zometa)를 추가하여 문화 매체 (CM)를 준비합니다. ALyS203 (셀 과학 기술 연구소) 또는 OpTmizer (Invitrogen) 미디어 γδ T 세포 (참조 6 9 자세한 정보)의 좋은 확장을 지원합니다. Zometa는 액체 형태 (4 mg/5-ml의 약병)에 제공됩니다. 5 μm의 솔루션을 준비하려면, 문화 매체의 30 ML에 Zometa 50 μl를 추가합니다. </li><li> Resuspend 문화 매체 세포 펠릿 및 1x10 6 세포 / ML을 조절할 수 있습니다. </li><li> 24 - 잘 플레이트의 각 우물에 1x10 6 세포를 포함하는 CM의 Pipet 1 ML. 대규모 문화, 전지 판 우물, 요리,이나 플라스크의 표면 영역에 따라이 0.5 X 10 6 세포 / cm에서 씨앗을하실 수 있습니다. </li><li> 그것은 문화의 볼륨 (24 잘 플레이트의 각 물론 100 μl)의 약 10 % 있도록 autologous 플라즈마 (섹션 1.3), 풀링 인간 AB 세라, 또는 FCS를 추가합니다. humidified 37 접시를 놓고 ° C 5 % 24-48 시간에 대한 CO 2 배양기. </li><li> 0.5-2 X 10 6 세포 / ML의 세포 밀도의 문화를 유지합니다. 신선한 매체마다 2-3일 인간 IL - 2 (1000 IU / ML) 전용 (Zometa없이)이 포함된 추가하고, 세포 증식의 정도 (그림 3)에 의하면, 새로운 우물하거나 필요한만큼 flasks으로 교양 세포를 전송합니다. 공급 플라즈마 또는 혈청 농도가 적어도 1% 유지 수 있도록 매체 혈청. </li><li> 일 12-14과 주파수, 표현형 및 유동세포계측법에 의해 γδ T 세포 (아래 참조)의 기능을 결정에 수확 세포. </li></ol> 3. 유동세포계측법에 의해 Phenotypic 분석 <ol><li> 형광 - 활성화된 셀 정렬 (외과) 튜브 2 X 10 5 세포를 포함하는 200 μl 샘플을 전송합니다. </li><li> 차가운 PBS 2 ML 추가 400 X g에서 5 분 원심 분리기. 그런 다음, 50 μl 외과 버퍼 (PBS + 1% FCS + 0.1 %의 나트륨 azide)에 알약을 resuspend. 샘플 (단클론 항체가 표 1에 나와 있습니다) 각 항체의 5 μl를 추가합니다. </li><li> 20 분 동안 짙은 어둠 속에서 얼음 품어. </li><li> 각 샘플 2 ML의 외과 버퍼를 추가한 다음 소용돌이. 4 400 X g ° C.에서 5 분 원심 분리기 샘플 조심스럽게 뜨는을 가만히 따르다. </li><li> 300 μl 외과 버퍼와 와동에있는 세포를 Resuspend. 흐름 cytometer (그림 2)에서 샘플을 분석할 수 있습니다. </li></ol> 4. IFN - γ 생산 분석 10 <ol><li> 분석 전날, RPMI 1640 미디어 플러스 Zometa (5 μm의) (이하 Z - Daudi 지정)과 하룻밤 10% FCS (RPMI - 10)에 culturing 3-5 X10 5 Daudi 세포 / ML로 자극기 전지를 준비합니다. </li><li> 2x10 6 셀 / ML에서 RPMI - 10 Z - Daudi 및 resuspend를 수집합니다. 둥근 바닥 96 - 웰 플레이트의 각 잘하는 Z - Daudi (2x10 5) 100 μl를 추가합니다. </li><li> RPMI - 10은 20 μg / ML에서 Brefeldin를 포함에 2x10 6 셀 / ML에서 γδ T 세포를 준비합니다. 각 잘 포함 Z - Daudi 전지에 γδ T 세포 현탁액 (2x10 5) 100 μl를 전송하거나 우물 (100 단 RPMI - 10 μl, 또는 phorbol 20 NG / ML 12 - myristate 13 - 아세테이트와 RPMI - 10 제어[PMA] 플러스 2 μg / ionomycin의 ML). </li><li> 몇 시간을 아래와 pipetting하여 섞는다. 37 ° C 5 % CO 2 배양기에서 4 시간을위한 부화. </li><li> 4 ° C에서 400 X g에서 5 분 접시를 원심 차가운 PBS 200 μl의 알약을 resuspend. </li><li> 외과 튜브에 샘플을 전송합니다. 차가운 PBS 4 ML을 추가하고 4 400 X g에서 5 분 튜브를 원심 ° C. </li><li> FITC - 복합 백신 TCRVγ9 (5 μl)와 PE/Cy5-conjugated 안티 인 경우에는 3 번 CD mAb (2.5 μl)와 외과 버퍼의 50 μl의 알약을 Resuspend. 부화는 실온에서 15 분 빛으로부터 보호. </li><li> IntraPrep 시약 1 100 μl를 추가하고 실온에서 15 분 알을 품다. 상온에서 400 X g에서 5 분 각 튜브와 원심 분리기에 PBS 4 ML을 추가합니다. </li><li> 흡인에 의해 표면에 뜨는를 제거하고 IntraPrep 시약 2 100 μl를 추가합니다. 떨지 않고 상온에서 5 분 알을 품다. </li><li> 테스트 튜브에 PE - 복합 백신 IFN - γ mAb 5 μl를 추가합니다. 부화는 실온에서 15 분 빛으로부터 보호. </li><li> 상온에서 400 X g에서 5 분 각 튜브와 원심 분리기에 PBS 4 ML을 추가합니다. 흡인에 의해 표면에 뜨는를 제거하고 외과 버퍼의 0.5 ML에있는 세포 펠렛을 resuspend. </li><li> 흐름 cytometer에 의한 세포를 분석할 수 있습니다. 인 경우에는 3 번 CD + TCRVγ9 + 세포에서 IFN 게이트 - γ (그림 4)의 표현을 확인합니다. </li></ol> 5. 대표 결과 : 문화의 개시에 PBMC에서 γδ T 세포의 비율을 결정하는 것이 중요합니다. 마찬가지로 그림에 표시됩니다. 2 A, 인 경우에는 3 번 CD + TCRVγ9의 비율은 + PBMC에서 γδ T 세포는 하루에 0에 1.6 %되었습니다. 중앙 메모리 phenotypes - 지배 집단은 CD27 + CD45RA + 순진한 또는 CD27 + CD45RA했다. γδ T 세포가 효율적으로 자극되었을 때, 그들은 (그림 3 A와 B) 일 3-5에 클러스터를 형성. 클러스터 형성이 지연되었을 때, 같은 다른 세포 유형의 성장 CD4 + 또는 CD8 + αβ T 세포 또는 NK 세포가 γδ T 세포 (그림 3 C 및 D)의 성장을 지배 수 있습니다. 문화 날로부터 14 일 후, γδ T 세포의 빈도가 성공적으로 γδ T 세포 배양 (그림 2 E)에서 교양 세포의 이상이 93.8 %로 증가했다. 교양 γδ T 세포 upregulated NKG2D 및 CD69 표현 (그림 2 G 및 H). 그들은 CD27 표시 - CD45RA - 효과기 메모리 표현형 (그림 2 F). γδ ​​T 세포의 기능이 시토킨 생산 및 세포 독성에 관한 평가했다. 세포내 IFN - γ 얼룩 γδ T 세포는 PMA / ionomycin 치료 또는 Z - Daudi 세포에 대한 응답으로 IFN - γ를 생산 것을 입증되는 zoledronate 치료 후 누적된 IPP (그림 4). 이러한 결과는 zoledronate 효율적으로 자극하고 기능 γδ T 세포를 확장할 수 있음을 나타냅니다. <table border="1"><tbody><tr><td> 튜브 </td><td> FITC </td><td> PE </td><td> ECD </td><td> PE/Cy5 </td></tr><tr><td> 1 </td><td> 인 경우에는 3 번 CD에 들어 </td><td> CD19 </td><td> CD45 </td><td> CD14 </td></tr><tr><td> 2 </td><td> 인 경우에는 3 번 CD에 들어 </td><td> TCRαβ </td><td> CD4 </td><td> CD8 </td></tr><tr><td> 3 </td><td> 인 경우에는 3 번 CD에 들어 </td><td></td><td></td><td> CD56 </td></tr><tr><td> 4 </td><td> TCR Vγ9 </td><td> TCRαβ </td><td> CD45 </td><td> 인 경우에는 3 번 CD에 들어 </td></tr><tr><td> 5 </td><td> TCR Vγ9 </td><td> NKG2D </td><td></td><td></td></tr><tr><td> 6 </td><td> TCR Vγ9 </td><td> CD69 </td><td></td><td></td></tr><tr><td> 7 </td><td> TCR Vγ9 </td><td> 마우스 IgG1 </td><td></td><td></td></tr><tr><td> 8 </td><td> TCR Vγ9 </td><td></td><td> CD45RA </td><td> CD27 </td></tr><tr><td> 9 </td><td> TCR Vγ9 </td><td></td><td> 마우스 IgG1 </td><td> 마우스 IgG1 </td></tr></tbody></table> 표 1. γδ T 세포의 여러 가지 빛깔의의 얼룩에 사용되는 단클론 항체. 우리 연구실에서 수행 γδ T 세포의 phenotypic 분석 예제는 그림에 표시됩니다. 2. <img src="/files/ftp_upload/3182/3182fig1.jpg" alt="그림 1"/> 그림 1. PBMC의 분리. 블러드 (7.5-8.0 ML)은 BD Vacutainer CPT 셀 준비 튜브 나트륨 헤파린과에 직접 그려 1,800 X g에서 20 분 centrifuged입니다. 원심 분리 후, 같은 결과 레이어는 처음부터 보로 볼 수ttom :) 플라즈마 - B) PBMC 및 혈소판 - C) 밀도 솔루션 - D) 폴리에스터 겔 - E) Granulocytes - F) 적혈구. <img src="/files/ftp_upload/3182/3182fig2.jpg" alt="그림 2"/> 그림 2. γδ T 세포의 일반적인 표면 표현형. PBMC는 14 일 동안 zoledronate 및 IL - 2와 함께 자극했다. 세포와 스테인드되었습니다 FITC - 라벨 안티 TCR γδ T Vγ9 및 세포 (A와 E)의 확장을 모니터하는 안티 - 인 경우에는 3 번 CD에 들어 PE/Cy5-labeled. γδ ​​T 세포는 TCRVγ9 자신의 표현으로 식별되었고, CD27 및 CD45RA (B와 F), NKG2D (C와 G), 또는 CD69 (D 및 H) 자신의 표현을 조사했다. <img src="/files/ftp_upload/3182/3182fig3.jpg" alt="그림 3"/> 그림 3. 대표 γδ T 세포 문화. PBMC는 IL - 2 (1000 IU / ML) 및 zoledronate (5 μm의)와 자극했다. 대표 필드 (IX71 거꾸로 현미경 [올림푸스] X 200) 표시됩니다. γδ ​​T 세포의 클러스터 및 집계는 γδ T 세포가 성공적으로 확장되었습니다 3 일 (A)과 5 일 (B)에 볼 수 있습니다. γδ ​​T 세포 성장 (C 및 D) 충분하지 때 대조적으로, 어떤 클러스터 또는 집계는 관찰되지 않았습니다. <img src="/files/ftp_upload/3182/3182fig4.jpg" alt="그림 4"/> 그림 4. IFN - γ 생산. γδ ​​T 세포는 RPMI - 10 (A) 또는 PMA / ionomycin (B) 또는 4 시간에 대한 Z - Daudi (C)와 incubated했다. 첫째, TCRVγ9의 표면 표현은 스테인드했습니다 그리고 IFN - γ 생산 세포내 IFN - γ 염색법에 의해 조사되었다. <img src="/files/ftp_upload/3182/3182fig5.jpg" alt="그림 5"/> 그림 5. γδ T 세포 문화의 속도론. 교양 세포 (A) 절대 번호, (B) γδ T 세포, 그리고 지정된 시간 지점에서 γδ T 세포 (C) 절대 번호의 비율입니다.

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+T+cell+receptor+&#947;&#948;+cells+recognize+endogenous+mevalonate+metabolites+in+tumor+cells.">Human T cell receptor &#947;&#948; cells recognize endogenous mevalonate metabolites in tumor cells.</a> J Exp Med. 197, 163-168 (2003).</li><li>Kabelitz, D., Wesch, D., He, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+of+gammadelta+T+cells+in+tumor+immunology.">Perspectives of gammadelta T cells in tumor immunology.</a> Cancer Res. 67, 5-8 (2007).</li><li>Roelofs, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+blood+monocytes+are+responsible+for+gammadelta+T+cell+activation+induced+by+zoledronic+acid+through+accumulation+of+IPP/DMAPP.">Peripheral blood monocytes are responsible for gammadelta T cell activation induced by zoledronic acid through accumulation of IPP/DMAPP.</a> Br J Haematol. 144, 245-250 (2009).</li><li>Dieli, F. <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+&#947;&#948;+T-lymphocyte+effector+functions+by+bisphosphonate+zoledronic+acid+in+cancer+patients+in+vivo.">Induction of &#947;&#948; T-lymphocyte effector functions by bisphosphonate zoledronic acid in cancer patients in vivo.</a> Blood. 102, 2310-2311 (2003).</li><li>Kondo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zoledronate+facilitates+large-scale+ex+vivo+expansion+of+functional+&#947;&#948;+T+cells+from+cancer+patients+for+use+in+adoptive+immunotherapy.">Zoledronate facilitates large-scale ex vivo expansion of functional &#947;&#948; T cells from cancer patients for use in adoptive immunotherapy.</a> Cytotherapy. 10, 842-856 (2008).</li><li>Espinosa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+synthesis+and+biological+activity+of+bromohydrin+pyrophosphate,+a+potent+stimulator+of+human+&#947;&#948;+T+cells.">Chemical synthesis and biological activity of bromohydrin pyrophosphate, a potent stimulator of human &#947;&#948; T cells.</a> J Biol Chem. 276, 18337-18344 (2001).</li><li>Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+profile+and+anti-tumor+effects+of+adoptive+immunotherapy+using+&#947;&#948;+T+cells+against+advanced+renal+cell+carcinoma:+a+pilot+study.">Safety profile and anti-tumor effects of adoptive immunotherapy using &#947;&#948; T cells against advanced renal cell carcinoma: a pilot study.</a> Cancer Immunol Immunother. 56, 469-476 (2007).</li><li>Murali-Krishna, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+antigen-specific+CD8+T+cells:+a+reevaluation+of+bystander+activation+during+viral+infection.">Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection.</a> Immunity. 8, 177-187 (1998).</li><li>Sato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+culture+medium+on+the+expansion+of+T+cells+for+immunotherapy.">Impact of culture medium on the expansion of T cells for immunotherapy.</a> Cytotherapy. 11, 936-946 (2009).</li><li>Abe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+immunological+evaluation+of+zoledronate-activated+V&#947;9+&#947;&#948;T-cell-based+immunotherapy+for+patients+with+multiple+myeloma.">Clinical and immunological evaluation of zoledronate-activated V&#947;9 &#947;&#948;T-cell-based immunotherapy for patients with multiple myeloma.</a> Exp Hematol. 37, 956-968 (2009).</li><li>Nakajima, J. <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+study+of+adoptive+immunotherapy+for+recurrent+non-small-cell+lung+cancer+patients+with+autologous+&#947;&#948;+T+cells.">A phase I study of adoptive immunotherapy for recurrent non-small-cell lung cancer patients with autologous &#947;&#948; T cells.</a> Eur J Cardiothorac Surg. 37, 1191-1197 (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

여기서 제시하는 방법은 PBMC에서 γδ T 세포의 효율적인 확장이 가능합니다. γδ ​​T 세포가 활성화되고 확대 zoledronate로와 IL - 2는 시토킨 생산 및 세포 독성에 의해 반사 완료 효과기 기능을 개발할 수 있습니다. 그것은보고되었음을 합성 phosphoantigens의 bromohydrin 나트륨 (BrHPP) 및 2 - 메틸 - 3 - butenyl - 1 - 나트륨 (2M3B1PP)는 또한 γδ T 세포를 확장하고 있지만, 그들은 상업적으로 사용할 수 없습니다....

<table border="1"><tbody><tr><th> 시약 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> ZOMETA </td><td> 노바티스 제약 K. K </td><td></td><td> zoledronate </td></tr><tr><td> PROLEUKIN </td><td> 노바티스 제약 </td><td></td><td> 인간 재조합 IL - 2 </td></tr><tr><td> BD Vacutainer CPT 셀 준비 튜브 나트륨 헤파린과 함께 </td><td> BD </td><td> 362753 </td><td></td></tr><tr><td> RPMI1640 </td><td> Invitrogen </td><td> 21870-076 </td><td></td></tr><tr><td> ALyS203 - 중간 </td><td> 셀 과학 기술 연구소 </td><td> 0301-7 </td><td></td></tr><tr><td> OpTmizer </td><td> Invitrogen </td><td> 0080022SA </td><td></td></tr><tr><td> brefeldin </td><td> 시그마 </td><td> B5936 - 200UL </td><td></td></tr><tr><td> phorbol 12 - myristate 13 - 아세테이트 (PMA) </td><td> 시그마 </td><td> P1585 - 1MG </td><td></td></tr><tr><td> ionomycin </td><td> 시그마 </td><td> 13909 - 1ML </td><td></td></tr><tr><td> IntraPrep </td><td> 베크맨 쿨터 </td><td> A07803 </td><td></td></tr><tr><td> 안티 - 인간 인 경우에는 3 번 CD - FITC 또는 PE/Cy5 </td><td> 베크맨 쿨터 </td><td> A07746 FITC A07749 PE/Cy5 </td><td></td></tr><tr><td> 안티 - 인간 CD4 - ECD </td><td> 베크맨 쿨터 </td><td> 6,604,727 </td><td></td></tr><tr><td> 안티 - 인간 CD8-PE/Cy5 </td><td> 베크맨 쿨터 </td><td> 6,607,011 </td><td></td></tr><tr><td> 안티 - 인간 CD14-PE/Cy5 </td><td> 베크맨 쿨터 </td><td> A07765 </td><td></td></tr><tr><td> 안티 - 인간 CD19 - PE </td><td> 베크맨 쿨터 </td><td> A07769 </td><td></td></tr><tr><td> 안티 - 인간 CD45 - ECD </td><td> 베크맨 쿨터 </td><td> A07784 </td><td></td></tr><tr><td> 안티 - 인간 CD56-PE/Cy5 </td><td> 베크맨 쿨터 </td><td> A07789 </td><td></td></tr><tr><td> 안티 - 인간 TCRαβ - PE </td><td> 베크맨 쿨터 </td><td> A39499 </td><td></td></tr><tr><td> 안티 인간 TCR - FITC Vγ9 </td><td> 베크맨 쿨터 </td><td> IM1463 </td><td></td></tr><tr><td> 안티 - 인간 CD27-PE/Cy5 </td><td> 베크맨 쿨터 </td><td> 6,607,107 </td><td></td></tr><tr><td> 안티 - 인간 CD45RA - ECD </td><td> 베크맨 쿨터 </td><td> IM2711 </td><td></td></tr><tr><td> 안티 - 인간 CD69 - PE </td><td> BD </td><td> 555531 </td><td></td></tr><tr><td> 안티 - 인간 NKG2D - PE </td><td> 베크맨 쿨터 </td><td> A08934 </td><td></td></tr><tr><td> 안티 humal IFNγ - PE </td><td> 베크맨 쿨터 </td><td> IM2717U </td><td></td></tr><tr><td> 마우스 IgG1 isotype 제어 PE </td><td> 베크맨 쿨터 </td><td> A07796 </td><td></td></tr><tr><td> 마우스 IgG1 isotype 제어 ECD 또는 PE/Cy5 </td><td> 베크맨 쿨터 </td><td> A07797 A07798 </td><td></td></tr></tbody></table>

expansion of human peripheral blood  &gamma;&delta; t cells using zoledronate

인간 γδ T 세포가 인식하고 스트레스 유발 항원의 다양한 반응, 따라서 본래 광범위한 안티 종양 및 안티 infective 활동을 개발하고 있습니다. 말초혈에서 γδ T 세포의 1 대부분 Vγ9Vδ2 T 세포 수용체 있습니다. 이러한 세포의 주요 histocompatibility 복합 독립적인 방식으로 항원을 인식하고 강한 cytolytic과 Th1과 같은 이펙터 기능을 개발할 수 있습니다. 1 따라서, γδ T 세포가 암 immunotherapy에 대한 매력적인 후보 효과기 세포 수 있습니다. Vγ9Vδ2 T 세포 같은 phosphoantigens에 대응 (E) -4 - 히드록시 -3 - 메틸 -하지만 - 2 - enyl 나트륨 (HMBPP), isoprenoid 생합성 통해 박테리아에 합성하는, 생산 2 isopentenyl 나트륨 (IPP), mevalonate 경로 3. 생리적 조건을 통해 진핵 세포에서 nontransformed 셀에 IPP의 세대 γδ T 세포의 활성화를 위해 충분하지 않습니다. 종양 세포에서 mevalonate 경로의 Dysregulation는 IPP의 축적과 γδ T 세포의 활성화로 연결됩니다. 3 aminobisphosphonates (예 : pamidronate 또는 zoledronate 등) farnesyl 나트륨의 synthase (FPPS), mevalonate 경로에서 IPP의 하류를 연기 효소의 세포내 수준을 억제하기 때문에 IPP 및 γδ T 세포 인식에 sensitibity는 therapeutically aminobisphosphonates 증가 수 있습니다. IPP 축적 aminobisphosphonates와 γδ T 세포를 활성화하여 암 우리에게 immunotherapy 수 aminobisphosphonates의 pharmacologically 관련 농도와 종양 세포보다 nontransfomred 세포에 덜 효율적이다. PBMC는 aminobisphosphonates로 치료하는 경우 4 흥미롭게도, IPP 때문에 효율의 monocytes에 쌓입니다 이러한 세포로 약물 이해. IPP 항원 제시 세포되고 주변 혈액에 Vγ9Vδ2 T 세포를 자극 축적 5 Monocytes가 6. 이러한 메커니즘을 바탕으로, 우리는 zoledronate와 인터루킨를 사용하여 γδ T 세포 문화의 대규모 확장을위한 기술을 개발 -2 (IL - 2). γδ T 세포의 확장 7 다른 방법은 합성 phosphoantigens의 bromohydrin 나트륨 (BrHPP) 8 2 - 메틸 - 3 - butenyl - 1 - 나트륨 (2M3B1PP)를 활용 9. 이러한 방법 중 모든 전 수 입양 immunotherapy에 사용하기 위해 γδ T 세포의 다수의 결과로 생체내 확장. 단, zoledronate은 FDA의 승인을 상용 시약입니다. CD45RA - - Zoledronate - 확장 γδ T 세포는 CD27 표시 효과기 메모리 표현형과 그들의 기능은 IFN - γ 생산 분석 7 평가할 수 있습니다.

Watch this Scientific Journal Video about Expansion of Human Peripheral Blood  ÃŽÂ³ÃŽÂ´ T Cells using Zoledronate at JoVE.com

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

주변 혈액 mononuclear 세포 (PBMC)에서 γδ T 세포를 확장하는 방법을 설명합니다. PBMC - 파생 γδ T 세포는 자극과 zoledronate과 인터루킨 - 2 (IL - 2)를 사용 확장됩니다. γδ ​​T 세포의 대규모 확장이 암의 autologous 세포 immunotherapy에 적용할 수 있습니다.

Zoledronate를 사용하는 사람 말초혈의 확장 γδ T 세포

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

expansion-of-human-peripheral-blood-t-cells-using-zoledronate

Research

JoVE Journal

Immunology and Infection

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

27.0K 조회수.  University of Tokyo Hospital. 주변 혈액 mononuclear 세포 (PBMC)에서 γδ T 세포를 확장하는 방법을 설명합니다. PBMC - 파생 γδ T 세포는 자극과 zoledronate과 인터루킨 - 2 (IL - 2)를 사용 확장됩니다. γδ ​​T 세포의 대규모 확장이 암의 autologous 세포 immunotherapy에 적용할 수 있습니다.

비디오: Expansion of Human Peripheral Blood  γδ T Cells using Zoledronate

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

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

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

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Bryostatin의 1/Ionomycin에 의해 종양 반응 T 세포의 생체내 전 확장 및 일반 감마 체인 크린 시토킨 제제

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

연구

면역학 및 감염병학

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

인간의 맨 틀 세포 림프 종의이 종이 식 마우스 모델에서 종양 Engraftment

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

암 연구학

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

말 초 혈액 Monocytes 유 방 암 세포 라인 공동 경작에서 Osteoclastogenesis의 인간의 전 임상 모델의 개발

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells 

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

확장, 정화, 그리고 사람 말초혈 NK 세포 기능 평가

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

인간 말초 혈액 단핵 세포 (PBMC) 번역 면역 종양학에 대한 인간화 제종이식 모델 (I-O) 연구

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (&#947;&#948;) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure &#947;&#948; T cells drug product for first-in-human use of allogeneic &#947;&#948; T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. 
The lymphocyte-rich fraction is utilized, and the &#947;&#948; T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of &#947;&#948; T cells, a clinical-grade magnetic cell-separation device and TCR&#945;&#946; beads are used to deplete contaminating T-cell receptor (TCR)&#945;&#946; T cells. The highly enriched &#947;&#948; T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched &#947;&#948; T cells in co-culture with the aAPCs facilitates the manufacture of highly pure &#947;&#948; T cells with an average fold expansion of &gt;229,000-fold from healthy donor blood.

Expansion and Enrichment of Gamma-Delta &#947;&#948; T Cells from Apheresed Human Product

Presented is a protocol for the expansion of Gamma-Delta (&#947;&#948;) T cell drug product. Lymphocytes are isolated by elutriation and &#947;&#948;-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The &#947;&#948; cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

감마 델타(γδ) T 세포의 확장 및 농축

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

소설<em&gt; 생체</em&gt; 유전자 전달 기술과<em&gt; 시험관</em근골격계 질환의 뼈 손실의 연구를위한&gt; 셀 기반 분석 실험

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

의학

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

디지털 PCR을 사용하여 마우스 조직에서 인간 T 세포의 Xegeneic Murine 모델 및 정량화에서 이식편 대 숙주 질환의 유도 및 채점

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

이식대 숙주 질환에서 수지상 세포의 역할을 조사하는 골수 이식 플랫폼

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

<meta charset="utf8"></head><body>방사선 조사된 마우스에서 효과적인 HSCT를 위한 골오가노이드 유래 세포

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

<meta charset="utf8"></head><body>치료용 조혈 줄기세포/전구세포를 채취하기 위한 In vivo Osteo-organoid 접근법

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

치료용 조혈 줄기세포/전구세포를 채취하기 위한 In vivo Osteo-organoid 접근법

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...