이 작품은 단 L. 던컨 공동 연구 기금 (CMB와 EJS), 국립 심장, 폐, 및 혈액 연구소 (US4HL081007), 백혈병 및 림프종 학회 임상 연구 학술 상 (CMB) 및 국립 암 연구소에 의해 지원되었다 (RO1 CA06150816, EJS).

1. Mononuclear 세포 절연 (일 0) <ol><li> 탯줄 혈액 단위의 20 % 분수의 출구 포트에 여성 luer 어댑터의 스파이크를 삽입하고, 주사기를 연결하고 피를 제거합니다. 50 ML의 원심 분리기 튜브에 따뜻하게 RPMI의 20 ML에 해동 혈액을 전송합니다. RPMI의 5 ML과 탯줄 혈액 가방을 헹굼과 동일한 원심 분리기 튜브로 전송할 수 있습니다. </li><li> 400 X g에서 10 분 동안 세포를 원심 분리기. 표면에 뜨는을 대기음. </li><li> 따뜻한 RPMI의 20 ML에 세포를 Resuspend. 50 ML의 원심 분리기 튜브의 Lymphoprep의 15 ML로 레이어 세포. 40분 @ 400 X g을 원심 분리기. </li><li> mononuclear 세포를 포함하는 인터페이스를 수확하고 RPMI의 20 ML에 씻는다. 십분 @ 450는 x g 용 원심 분리기. </li><li> 표면에 뜨는을 기음과 RPMI의 20 ML에 씻는다. 세포를 세어 5 분 @ 400 X g에 스핀. </li><li> 5시 표면에 뜨는 및 resuspend 세포를 대기음 X 10 6 mononuclear 세포 / Cellgenix DC 미디어 (혈청 무료)의 ML. EBV-LCL 세대 1 ML를 제거하고 15 ML CE에 투입관을 ntrifuge. </li></ol> 2. 수지상 세포 생성 (일 0에 시작) <ol><li> 세포의 판 2 ML은 당 잘 6 잘 판에 (5 X 10 6 세포 / DC의 미디어 ML에 이미 있습니다.) 1-2시간에 대한 보육에 둡니다. </li><li> 1-2시간 후, PBS로 3-4 회 세척하여 비 점착성의 세포를 씻어. 비 점착성의 세포와 동결을 수집합니다. 2 ML / 잘 1000 U / ML IL-4 및 800 U / ML GM-CSF를 포함하는 DC 미디어를 추가 할 수 있습니다. </li><li> 3-4일 DC 개시 후, 100을 추가 μL / 우물 1000U/mL IL-4 및 800 U / ML GM-CSF의 최종 농도를 포함하는 미디어에 의해 IL-4 및 GM-CSF를 보충. </li><li> 일 5-6, 수확 전송 피펫으로 잘를 스크랩하여 DC에. 오분 @ 400 X g의 큰 세포와 원심 분리기를 계산합니다. 2x10 6 셀 / DC의 미디어 ML의 기음 미디어 및 resuspend DC. / 잘 24 잘 접시의 0.5 ML를 추가합니다. </li><li> 10 전염성 단위의 전부다 당 셀에 Ad5f35pp65 벡터를 사용하여 DC를 Transduce. vecto을 추가24 잘 플레이트의 각도에 연구. 1.5 시간에 세포를 배양. 1.5 시간 후, / 잘 DC 미디어 함유의 1.5 ML를 추가합니다 : 1000 U / ML IL-4, 800 U / ML GM-CSF, 10 NG / ML TNF-알파, 1 μg / ML PGE-1, 100 NG / ML IL-6, 10 NG / ML IL-1beta. 이러한 크린 시토 킨은 지구 코디네이터를 성숙. </li></ol> 3. EBV-LCL의 세대 (일 0에 시작) <ol><li> 5 X 10 6 mononuclear 세포를 포함하는 튜브를 원심 분리기. 농축 EBV B95.8 표면에 뜨는 200 μL하고 완벽한 미디어 1.8 ML (RPMI + 10% FBS + 2mM L-글루타민) 함유 시클로 스포린 (1 μg / ML)를 추가합니다. </li><li> 96 - 웰 플레이트의 10 우물, 5 우물에 200 μL에 세포의 나누어지는 100 μL. 세포의 단 100 μL를 포함하는 우물에 전체 미디어 + 시클로 스포린의 100 μL를 추가합니다. 멸균 물에 남아있는 우물을 입력합니다. </li><li> LCL 매주을 먹이고 ~ 2 주 후에는 24도 판에 96 - 웰 플레이트에서 확장합니다. 다른 주 후, T25 플라스크에 전송하고 다음 주 이전에에T75 플라스크입니다. 이 단계에서 귀하가 LCL의 aliquots를 고정하는 것이 좋습니다. 그것은 일반적으로 LCL의 충분한 숫자를 생성 할 수 1개월 소요됩니다. </li></ol> 4. CTL 개시 - 칠일 코디네이터의 개시 후 <ol><li> 30 GY에서 수확 DCS 및 비추다. 4 x와 수를 씻으십시오. 인간의 혈청 (45 % RPMI, 45% CLICK의, 10 % 인간의 혈청, 2mM L-글루타민) @ 1 × 10 5 DCS / ML.를 포함하는 T 세포의 미디어에 Resuspend DC </li><li> 단계 2.2의 비 점착성의 세포를 해동. 세포를 씻어하고 계산합니다. 2 X 10 6 비 점착성의 세포 / 인간의 혈청을 포함하는 T 세포의 미디어 ML에 Resuspend. 10 NG / ML IL-7, IL-12, 5 NG / ML IL-15을 추가합니다. 한 셀의 ML 당 잘 24 잘 판에 추가 할 수 있습니다. 당 잘 DC의 1 ML을 추가합니다. 멸균 물과 24 잘 접시의 빈 우물을 입력합니다. </li><li> 칠일 T 세포의 개시 후, 피드 및 / 또는 인간 혈청을 포함하는 T 세포 미디어와 세포를 분리. </li><li> LCL 준비가 될 때까지 9~12일 T 세포의 개시 후 T 세포를 동결. </li><li> OLCL은 확장하고 T75 플라스크에 passaged 아르 nce 5 분 @ 400 X g 용 centrifuging하여 LCL을 transduce. 나 역시 셀 100 전염성 단위의에서 세포 펠렛에 벡터를 추가합니다. 1.5 시간 품다. 후 5 × 10 5 LCL / ML에서 전체 미디어에 resuspend과 24 잘 판에 당 잘이 ML을 추가 1 시간 30 분, 12. </li><li> 이일 후, 40 GY에서 LCL과 비추다 수확. 4 x와 수를 씻으십시오. 2.5 × 10 5 세포 / ML에서 인간의 혈청을 포함하는 T 세포 미디어에서 LCL을 Resuspend. 한 LCL의 ML 당 잘 24 잘 플레이트를 추가합니다. 또한, Grex40 문화 장치에뿐만 아니라 5 NG / ML IL-15 5 X 10 6 LCL를 추가합니다. 13 </li><li> T 세포를 수확. @ 400 XG 5 분 원심 분리기는 표면에 뜨는을 대기음, 1 X 10 6 T 세포 / ML에서 인간의 혈청을 포함하는 T 세포의 미디어에 resuspend. 5 NG / ML IL-15을 추가하고 24 잘 판에 이미있는 LCL에 1 ML / 잘의 T 세포 (1 × 10 6 T 세포의 총)를 추가합니다. Grex40에서 가져30 ML에 미디어의 총 볼륨입니다. </li><li> 세포가 합류하는 경우 일 3-4 후 자극에 다음 (판)을 1:1으로 분할 50 U / ML IL-2를 포함하는 새로운 미디어를 추가 할 수 있습니다. 그들은 합류하지 않은 경우 다음 단순히 ½ 미디어를 대기음 50 U / ML IL-2를 포함하는 미디어로 교체하십시오. Grex40에서, 절반 미디어를 대기음 신선한 미디어로 교체, 50 U / ML IL-2를 추가합니다. </li><li> 일 7 단계 4.6에서와 같이 반복하되 IL-15, 자극의 일에 IL-2 사용하지 않을. </li></ol> 5. 대표 결과 GMP 준수 FDA 승인 제조 프로토콜의 개략도는 그림 1에 설명되어 있습니다. 이 과정은 약 50 일이 소요됩니다. 그러나, 코드 혈액은 바이러스 경험이 풍부한 T 세포 부족하고 따라서 우리는 주요 순진 T 세포의 예를 생체에 필요, 바이러스 특정 T 세포는 기존의 메모리 T 세포를 확대 생성의 전형적인 방법. 이렇게하려면 우리는 IL-12, 그리고 IL-15, 수지상 세포뿐만 아니라 크린 시토 킨 IL-7을 사용하여바이러스 특이성을 생성에 필요한. 3 stimulations 후, 수율은 100x10 6 셀 이상이어야합니다. 충분한 세포를 사용할 수없는 경우 CTLs의 원하는 번호를 사용할 수 있습니다 때까지 추가 stimulations을 수행 할 수 있습니다. 이 세포의 대부분은 CD4의 혼합물 +와 CD8 + T 세포와 3 번 CD +해야합니다. 이하 15 % 이상 NK 세포 (CD3-/CD56 +) 및 1 % 미만 CD19 + B 세포 (그림 2)이 있어야합니다. 확장 된 CTL은 아데노 바이러스에서 CMV, hexon 및 penton에서 항원 pp65을 인식뿐만 아니라, EBV-LCL에 표현 수많은 EBV의 항원해야합니다. ELISPOT 분석에서 테스트 할 때 CTL이 분비해야 더 IFN-? 관련이없는 항원 (그림 3)보다 이러한 항원에 대응 인치 CTL은 또한 바이러스 성 펩타이드 - 펄스 대상 한테 그런 PHA의 폭발을 lyse해야합니다. 51 크롬 자료 분석에서, CTL은 CMVpp65-펄스와 아데노 바이러스, LCL을 lyse해야 hexon 및 /또는 대상 penton - 펄스 수 있지만이 무관 한 펩티드 (그림 4)와 펄스 타겟팅 할 수 있습니다. <img src="/files/ftp_upload/3627/3627fig1.jpg" alt="1 그림."/> 그림 1. 탯줄 혈액에서 여러 바이러스에 특정한 CTL의 세대. 탯줄 혈액에서 CTL 제조의 모든 과정을 보여주는 설계도. 먼저 탯줄 혈액 mononuclear 세포는 fractionated 탯줄 혈액 단위의 20 % 분율으로부터 격리되어 있습니다. LCL 생성에 저장됩니다 5x10 6 세포를 제외한 모든 세포는 다음 비 점착성의 세포 수확 및 냉동되는 시점에, 1-2 시간 동안 수지상 세포 미디어에 도금되어 있습니다. DC 그런 다음 DC 미디어 IL-4 및 GM-CSF를 포함하는 공급하고 있습니다. 문화 5 일 후, DC는 성숙되고 immunodominant CMV pp65 항원을 포함하는 adenoviral 벡터와 transduced. 시작시,이 DC는 비 점착성의 세포뿐만 아니라 크린 시토 킨 IL-7, IL-12와 결합되어와 IL-15. 다음 stimulations에서 같은 adenoviral 벡터는 항원 제시 세포로 사용되는 EBV-LCL을 transduce하는 데 사용됩니다. IL-15은 두 번째 자극과 IL-2 이후에 사용됩니다. <img src="/files/ftp_upload/3627/3627fig2.jpg" alt="그림 2."/> 그림 2. T 세포를 결과의 표현형. 표시하면 림프구 문에 포함 된 라이브 세포의 비율입니다. CTL은 3 번 CD +와 CD4 + CD8 + 또는하지만 대부분 CD3-/CD56-이며 CD19 -. <img src="/files/ftp_upload/3627/3627fig3.jpg" alt="그림 3."/> 그림 3. T 세포 기능. T 세포 특이성은 IFN-γELISPOT에 의해 테스트되었습니다. T 세포는 중복 펩티드는 hexon, penton, pp65의 전체 단백질을 걸친으로 펄스과 관련이없는 항원 IE-1되었다. 혼자 CTL 혼자 미디어를 나타냅니다. Autologous LCL은 방사선 및 1x10 5 / 잘에 추가되었습니다. 표시하는 셀 C를 형성 평균 곳입니다세중의 우물의 ount. <img src="/files/ftp_upload/3627/3627fig4.jpg" alt="4 그림."/> 그림 4. CTL의 Cytolytic 활동. 바이러스 항원을 표현 대상을 lyse 할 수있는 결과 CTL의 능력은 51 크롬 분석에서 테스트되었습니다. 51 CR-라벨 Autologous PHA의 폭발이 중복되는 펩티드는 전체 항원 또는 51 LCL은 CTL과 cocultured 된 Autologous CR-라벨에 걸쳐와 펄스되었습니다. 4시간 후, 감마 자료는 감마 카운터에 계산되었습니다.

<ol>
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Hematol. 91, 588 (2010).</li><li>Einsele, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infusion+of+cytomegalovirus+(CMV)-specific+T+cells+for+the+treatment+of+CMV+infection+not+responding+to+antiviral+chemotherapy.">Infusion of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy.</a> Blood. 99, 3916 (2002).</li><li>Bao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+cytomegalovirus+pp65+and+IE-1+specific+cytotoxic+T+lymphocytes+for+cytomegalovirus-specific+immunotherapy+following+allogeneic+stem+cell+transplantation.">Expansion of cytomegalovirus pp65 and IE-1 specific cytotoxic T lymphocytes for cytomegalovirus-specific immunotherapy following allogeneic stem cell transplantation.</a> Biol. Blood Marrow Transplant. 14, 1156 (2008).</li><li>Shpall, E. J., Bollard, C. M., Brunstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+cord+blood+transplant+therapies.">Novel cord blood transplant therapies.</a> Biol. Blood Marrow Transplant. 17, S39-S45 (2011).</li><li>Vera, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+production+of+antigen-specific+T+cells+for+preclinical+and+clinical+applications+using+gas-permeable+rapid+expansion+cultureware+(G-Rex.">Accelerated production of antigen-specific T cells for preclinical and clinical applications using gas-permeable rapid expansion cultureware (G-Rex.</a> J. Immunother. 33, 305 (2010).</li></ol>

저자는 공개 할 수 없어요.

<html> CBT 이후 바이러스 감염을 제어하기위한 현재의 전략은 효과적 일 수 있지만, 그들은 상당한 toxicities와 관련된, 비싸고, 나중에 감염에 대한 장기적인 보호를 부여하지 않습니다. 사실, 일부 바이러스 약물의 사용은 다른 보호 될 바이러스 특정 T 세포의 확장을 제한 할 수 있습니다. 14 또 다른 옵션은 기증자 파생 바이러스 특정 T 세포의 주입이다. 우리와 다른 사람들이 이러한 T 세포?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 21870-076 </td></tr><tr><td> DC 미디어 </td><td> CellGenix </td><td> 20801-0500 </td></tr><tr><td> EHAA (클릭의 중간) </td><td> 어바인 과학 </td><td> 9195 </td></tr><tr><td> 인간 혈청 </td><td> 쌍둥이 바이오 제품 </td><td> 100-110 </td></tr><tr><td> 가스 투과성 Cultureware 18 </td><td> 윌슨 - 늑대 </td><td> 80040S </td></tr><tr><td> IL-2 </td><td> Chiron (TCH 약국) </td><td></td></tr><tr><td> IL-12 </td><td> NCI / CTEP </td><td></td></tr><tr><td> IL-15 </td><td> CellGenix </td><td> 1013-050 </td></tr><tr><td> IL-7 </td><td> R &amp; D </td><td> AFL207 </td></tr><tr><td> IL-1beta </td><td> R &amp; D </td><td> AFL201 </td></tr><tr><td> IL-6 </td><td> 세포Genix </td><td> 1004-050 </td></tr><tr><td> GM-CSF </td><td> TCH 약국 </td><td></td></tr><tr><td> IL-4 </td><td> R &amp; D </td><td> <a href="http://www.rndsystems.com/pdf/AFL204.pdf" target="_blank">AFL204</a> </td></tr><tr><td> TNF-알파 </td><td> R &amp; D </td><td> AFL210 </td></tr><tr><td> Ad5f35pp65 </td><td> BCM CAGT 벡터 생산 시설 </td><td></td></tr><tr><td> 플라즈마 전송은 여성 luer 어댑터 설정 </td><td> 헌장 의료 </td><td> 89-550 - 66j </td></tr><tr><td> Lymphoprep </td><td> Nycomed </td><td> 1114550 </td></tr></tbody></table>

expanding cytotoxic t lymphocytes from umbilical cord blood that target cytomegalovirus, epstein-barr virus, and adenovirus

줄기 세포 이식 후 바이러스 감염, 특히 탯줄 혈액 후, 죽음의 가장 일반적인 원인 중 일부입니다 (CB) 이식 (CBT) 어디 CB가 감염에서 수신자를 보호 할 수 있습니다 바이러스 경험이 풍부한 T 세포 1 감지 숫자가 포함되어 있지 않습니다. - 우리와 다른 사람들이 바이러스에 특정한 CTL seropositive 기증자에서 생성하고받는 사람에게 주입하는 것으로 나타났습니다 4 안전하고 보호합니다. 5-8는하지만, 최근까지 바이러스 특정 T 세포가로 인해 가능성, 탯줄 혈액에서 생성 할 수 없습니다 바이러스 특정 메모리 T 세포의 부재. 더 나은 나이브 T 세포의 생체 프라이밍 조건에서을 모방하기 위해, 우리는 따라서 T 세포 특이성 운전, CB-파생 수지상 세포 (DC)은 immunodominant CMV의 항원 pp65을 포함하는 adenoviral 벡터 (Ad5f35pp65)과 transduced 사용 방법을 설립 시작시 CMV와 아데노 바이러스 9.을 향해, 우리는 이러한 mA를 사용tured 코디네이터뿐만 아니라 모두를 표현 IL-7, IL-12와 IL-15. 두 번째 우리가 EBV-변환 B 세포를 사용 자극, 또는 EBV-LCL, 10 크린 시토 킨의 존재에 CB-파생 T 세포 잠재 및 lytic EBV의 항원. Ad5f35pp65 - transduced EBV-LCL은 두 번째 자극의 IL-15의 존재에 T 세포를 자극하는 데 사용됩니다. 이후 stimulations는 EBV-LCL 및 IL-2 Ad5f35pp65 - transduced을 사용합니다. 50x10 6 CB mononuclear 세포부터 우리는 antigenic의 자극에 대한 응답으로 항원 펄스 목표 및 출시 크린 시토 킨을 lyse 150 X 10 6 바이러스 특정 T 세포의 이상을 생성 할 수 있습니다. 11이 세포를 사용하여 GMP 호환 방식으로 제조 된 단 20 % fractionated 탯줄 혈액 단위의 분율하며 임상 사용하기 위해 번역되었습니다.

Watch this Scientific Journal Video about Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus at JoVE.com

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

여기가 첫 번째 좋은 생산 관행 (GMP)을 준수하는 탯줄 혈액, 주로 나이브 T 세포의 소스에서 바이러스 특정 세포 독성 T 림프구 (CTL)를 생산 방법을 설명합니다.

탯줄 혈액에서 세포 독성 T 림프구 확장이 대상 사이토 메갈로 바이러스, 엡스타인 - 바 바이러스, 그리고 아데노 바이러스

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

expanding-cytotoxic-t-lymphocytes-from-umbilical-cord-blood-that

Research

JoVE Journal

Immunology and Infection

12.7K 조회수.  Baylor College of Medicine. 여기가 첫 번째 좋은 생산 관행 (GMP)을 준수하는 탯줄 혈액, 주로 나이브 T 세포의 소스에서 바이러스 특정 세포 독성 T 림프구 (CTL)를 생산 방법을 설명합니다.

비디오: Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

준비 및 사용 HIV - 1 차 감염된 CD4 + 대상 세포로 T 세포 자연 킬러 세포 세포 독성 Assays에서

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

연구

면역학 및 감염병학

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

세포 독성 T Lymphocytes에 의해 베타 세포 죽음의 중재를 평가하는 방법

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

세균성 접착 연구에 전단 응력을 소개합니다

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

엡스타인 - 바 바이러스 성장 변화 Lymphoblastoid 전지 라인 구축

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

임상 신청<em&gt; 잠자는 숲속의 미녀</em유전자 주변과 배꼽 탯줄 혈액에서 T 세포를 수정하는 셀을 제시&gt;과 인공 항원

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

탯줄 혈액에서 전구체 B-세포 하위 집합의 절연

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

분리 및 나이브 CD4 T 림프구의 TH17 분화

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Development of an IFN-&amp;#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

제대혈 이식을 다음과 IFN-γ 평가하기 ELISPOT 분석을 수두 - 대상 포진 바이러스 - 특정 세포 성 면역의 개발

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

말초 혈액에서 인간 Alloantigen 특정 T 세포의 생성

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

레이디 얼 이동성 및 레트로 바이러스 복제 벡터 형질 도입, 비 접착 Alloresponsive T 림프구의 세포 독성 기능

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...