이 작품은 셀룰러 테라피 (계약 NIH - NHLBI (HB - 10 - 03) HHSN26820100000C) (CMR), 셀 기반의 치료를위한 전문 센터에 대한 생산 지원에 의해 지원됩니다 부여 NIH - NHLBI 1 U54 HL081007 (CMR), ASBMT 영 인베스티게이터 수상 (UG와 JV), 임상 연구 수상 (UG)에서 백혈병과 림프종 사회 특별 연구원, 그리고 에이미 Strelzer Manasevit 학술 수상 (AML).

1. 직류 nucleofection <ol><li> 셀 Genix IL4 (1000U/ml)와 보충 미디어 GMCSF (800IU/ml)와 추가 DC의 성숙의 크린 시토킨 IL4을 (1000U 사용하여 24 시간을위한 성숙을 사용하여 오일에 대한 교양 플라스틱 준수를 사용하여 풍부한되었습니다 하베스트 단핵구 - 파생 DC가, 3ml 전송 피펫과 부드러운 resuspension로 / ML), GMCSF (800IU/ml), IL6100ng/ml, TNF - α 10ng/ml, IL1 - β 10ng/ml 및 PGE2 (1μg/ml) 1. </li><li> trypan 파랑, 노 적은 6 0.5x10 이상과 배 15ml 튜브로 전송하고 더 이상 6 2x10 이상의 셀 / 튜브를 사용 가능한 DC가를 계산합니다. </li><li> 상업 중심지 @ 200g에 대한 DC가를 원심 분리기. 37 ° C / 5% CO 2 배양기에서 번호판을 처리 12 잘 조직 문화의 세 우물에 2ml/well -이 기간 동안 사전에 따뜻한 셀 Genix 미디어는 DC의 성숙의 크린 시토킨 (DC의 성숙 미디어)과 보완. </li><li> 일단 세포가 스피닝 완료, 뜨는을 기음과 DNA 5μg / 튜브의 최종 농도에 튜브의 각 관련 유전자 plasmids를 추가합니다. 이 경우에는 플라스미드 인코딩 튜브에 IE1 - pp65 # 1, 튜브에 Hexon - 펜톤 # 2, # 3 관 EBNA1 - LMP2 - BZLF1를 추가합니다. </li><li> Amaxa의 nucloefection 솔루션의 100μl와 Resuspend DCS 및 DNA가 잘 혼합하고 nucleofection의 cuvettes로 전송할 수 있습니다. </li><li> 4D nucleofector의 장소 cuvettes는, 프로그램 CB150 (Amaxa / Lonza)를 선택하고 시작을 누르십시오. </li><li> nucleofection가 쿠베트에 2ml 미리 예열 셀 Genix DC의 성숙 미디어의 500μl 추가 직후, prewarmed의 나머지 1.5 ML를 포함하는 준비된 12 잘 판에 2-3 시간, 전송 nucleofected DC가 아래로 pipetting과로 부드럽게 혼합 DC의 성숙 미디어. 37에 전송 ° C / 5% CO 2 배양기를 추가로 12 18hrs하십시오. </li></ol> 2. T 세포 자극 <ol><li> 수확 및 30Gy에서 nucleofected DC가를 계산하고, 비추다. CTL 매체 10ml (45 % RPMI, 45 % 클릭수 EHAA 10 % FBS, 2mM Glutamax)과 CTL 미디어 ML 당 resuspend @ 3 X 10 5 DC가 함께 한 번 씻으십시오. </li><li> 풀 7.5x10 5 (2.5ml)와 plasmids 각을 포함하는 DC가의 15x10 5 (5ml)의 최대 최소 그리고 보육에 배치됩니다 G - 렉스 장치에 DC가 풀링을 전송. </li><li> 응답 세포의 준비를 위해 이전에 냉동 PBMCs 또는 DC의 선택 (부착 또는 CD14 선택) 이후 유지 비 점착 성의 mononuclear 세포 중 하나를 사용합니다. 세포를 해동, prewarmed 문화 매체 전송, CTL 매체와 한 번 씻는다. CTL 미디어의 세포를 Resuspend, 세포를 포함하고 ML 당 2x10 6 세포의 농도로 끌고 오너라. 와 300ng IL7 (10ng/ml - 최종 CONC.) - 30000U IL4 (. 최종 CONC 1000U/ml)와 15x10 6 셀 또는 7.5ml 및 보충 가져가라. </li><li> G - 렉스와 30ml의 총 볼륨 CTL 매체와 생물 반응기까지 가기 PBMC의 7.5ml을 (15x10 6 셀) 전송합니다. </li><li> 문화 37 6~7일을위한 G - 렉스 ° C / 5% humidified CO 2 배양기. </li></ol> 3. T 세포 확장 <ol><li> 일 6-7 일, 미디어의 대기음의 10ml, 그때 10ml 피펫과 함께 미디어의 남은 20ml에 세포를 혼합하고 trypan 파랑을 사용 가능한 세포를 계산합니다. 있다면 &lt;50x10 6 새로운 미디어 + 크린 시토킨과 보충. &gt; 50x10 6 세포가있다면 세포 현탁액의 10ml를 제거, 새로운 G - 렉스로 전송하고 신선한 CTL 미디어 + 크린 시토킨과 함께 모두 G - 렉스스 먹이. </li><li> 추가 4-6일을위한 문화. 일단 충분한 세포는 나중에 사용하기 위해 CTL과 cryopreserve 초과 phenotypic과 기능 특성을 수행 확장되었습니다. </li></ol> 4. 대표 결과 : 우리의 FDA 승인 multivirus 특정 CTL 생성 과정의 개략도는 그림 1에 표시됩니다. 바이러스 반응 T 세포를 자극 adenovectors과 EBV - LCL를 사용하여 대회 multivirus CTL 프로토콜과는 달리 2 우리의 DNA plasmids과 전염성 바이러스 자료를 교체했습니다 그 바이러스 3 각에서 파생된 여러 항원을 인코딩. trivirus의 CTL을 자극하기 위해 우리는 EBV 세 multicistronic plasmids 인코딩 Hexon과 펜톤 아데노 바이러스의 IE1 및 pp65 CMV, 그리고 EBNA1, LMP2 및 BZLF1을 설계했습니다. 이러한 항원들은 우리 자신과 T 세포가 교수실 hexon과 펜톤 2,4-6에 대한 감독 및 CMV - 1E1과 CMV - pp65은 생체내 7 보호 아르에게 보여주는 다른 그룹의 임상 결과를 장려하고 기반으로 선정되었습니다. EBV 들어, EBNA1는 immunodominant CD4 + 모든 EBV - 관련 malignancies 및 일반 EBV에 감염된 B 세포 표현 T 세포의 대상 항원이다 8,9, LMP2 여러 HLA 유형에 걸쳐 immunogenic이며, 대부분의 EBV malignancies로 표현, 10,11 BZLF1 동안 대부분의 개인에서 모두 CD4 +와 CD8 + T 세포를 자극하고 공동에 대한 가능성이 중요합니다 immunodominant, 즉각적인 초기 lytic 순환 항원을 인코딩바이러스 12 복제 세포 ntrol. 추가로 우리의 제조 방법을 최적화하기 위해 우리는 CTL 자극 13,14에 대한 minimalized, 항생제없는 (FDA 호환) plasmids를 생성 자연 기술과 협력. 같은 IFNγ ELIspot에 의해 측정된 최적의 DNA plasmids에 대한 응답으로 바이러스 구체적인 T 세포의 빈도가 큰 것을 높은 세포 생존 (데이터가 표시되지 않음) 3. 그림 2 프로그램을 유지하면서이 전략을 사용하면 우리는 지속적으로&gt; 35%의 nucleofection 효율성을 달성 동일한 항원 (N = 8 아데노 바이러스, N = 4 CMV하고, N = 2 EBV)를 표현하는 기존 pShuttle 기반 표현 plasmids에 대한 응답으로보다. DC의 최적 비율 : R 비율 (N = 2 기증자) : PBMC는 1시 50분의 비율이 1시 20분 S에 비해 하위 최적 활성화를 생산 그림 3과 같이 강력한 T 세포 자극에 중요했다. 광범위한 항원 특이성과 충분한 CTL 숫자의 생산 세 바이러스에 대한 임상 효능을위한 필수 조건입니다. 문화 증가 IL4과 IL7의 덧셈으로 레퍼토리와 특이성이 그림 4B에 표시하면서 이것은, 종래의 24 잘 접시 (그림 4A) 15와 비교하여 우수한 T 세포 확장을 지원하는 G - 렉스에 CTL 문화에 의해 이루어집니다 CMV - pp65 - 파생 HLA - A2에 대한 반응 T 세포의 빈도가 resticted 어디 NLV 펩타이드는 IL4 및 / 또는 IL7 16,17의 존재 또는 부재에서 생성된 문화에 평가되었다. cryopreservation / 주입을위한 최종 제품에 우리가 흐름 cytometric 분석을 수행 확장 세포, 세포 시토킨 얼룩 / IFNγ ELIspot 및 CR 51 릴리스 assays의 표현형 및 기능적 능력을 평가합니다. 일반적으로 생성된 세포가 CD4 + 두 T 세포 구획에 감지 항원 특이성과 CD8 + T 세포의 혼합 인구 polyclonal 있습니다. CTL들은 생체내 (그림 5)에 이식 - 대 - 호스트 질병 (GvHD)을 유발해서는 안 나타냅니다, 바이러스 부분 - HLA 일치 목표 부정하지 바이러스성 항원 표현 대상 세포를 죽일하지만 수 있습니다. <img src="/files/ftp_upload/2736/2736fig1.jpg" alt="그림 1"/> 그림 1. rCTL ​​생성 프로토콜. 첫째, DC가이 바이러스 항원 - 인코딩 plasmids와 nucleofected 그리고 R에 au​​tologous PBMCs와 혼합 : 10 20시 1분의 S. 전지는 다음 수확 집계, 기능, 정체성과 불임에 대한 검사, 그리고 임상 사용을위한 cryopreserved IL4과 IL7의 면전에서 10~14일에 대한 G - 렉스로 확장됩니다. <img src="/files/ftp_upload/2736/2736fig2.jpg" alt="그림 2"/> 그림 2. 최적의 DNA plasmids은 체외에서 우수한 T 세포 활성화를 유도. DC가이 최적화, FDA 준수 plasmids 인코딩 Hexon와 펜톤 (교수실), IE1 및 pp65 (CMV) 및 EBNA1, LMP2 및 BZLF1 (EBV) 또는 동일한 항원을 인코딩 기존 pShuttle의 plasmids과 nucleofected되었습니다. 이들은 T 세포를 자극하는 데 사용되었습니다과 특이성은 IFNγ ELIspot 10 일 이후의 자극에 의​​해 분석되었다. <img src="/files/ftp_upload/2736/2736fig3.jpg" alt="그림 3"/> 그림 3 최적의 DC :. CTL 활성화를위한 T 세포의 비율. 이 기증자의 DC가 세 개 모두 최적화 plasmids와 nucleofected 후 1시 20분 또는 1시 50분 DC에서 autologous PBMCs을 자극하는 데 사용되었습니다 PBMC 비율. 활성화 T 세포의 빈도가 IFNγ에 의해 ELIspot 일 10 일 평가했다. <img src="/files/ftp_upload/2736/2736fig4.jpg" alt="그림 4"/> 그림 4. 향상 크린 시토킨를 사용하여 G - 렉스의 T 세포 확장. 패널 A는 G - 렉스 장치뿐만 아니라 현미경으로 평가 가스 침투성 막에 CTL 외관을 보여줍니다. 접시 VS G - 렉스를 취급 대회 조직 문화 달성 셀 출력 비교도 표시됩니다. 패널 B는 아니 시토킨 혼자 IL4, IL7 혼자하고 IL4 + IL7의 존재의 확장 문화에 달성 CMV pentamer 긍정적인 CTL의 빈도를 보여줍니다. <img src="/files/ftp_upload/2736/2736fig5.jpg" alt="그림 5"/> 그림 5. 표현형 및 확장 CTL의 기능을 수행합니다. 와 CD8 + - 패널 A는 CD4 + (도우미 45 %)의 혼합물로 polyclonal있는 확장 multivirus의 CTL의 표현형의 대표 예를 나타냅니다 (42 % - 세포 독성) 대부분 (95 %)을 표현하는 T 세포, 메모리 마커 CD45RO + / CD62L +. 패널 B는 이러한 세포가 모든 자극 항원에 대한 특정하고 IFNΓ의 생산과 항원 자극 후 TNFα를 감지하는 세포 시토킨 얼룩에 의해 평가로 polyfunctional 것을 보여줍니다. 패널 C는 확장 CTL가 측정으로 작동되는 보여줍니다 CR 51 분석에 의해. Autologous LCL, 단독으로 또는 CMV - pp65을 표현 NULL 벡터 또는 adenoviral 벡터와 transduced는 타르로 사용되었습니다도착. Alloreactivity은 대상으로 allogeneic PHA의 폭발을 사용하여 평가되었다.

<ol>
	<li>Kaka, A. S., Foster, A. E., Weiss, H. L., Rooney, C. M., Leen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+dendritic+cell+maturation+and+IL-12+producing+capacity+as+markers+of+function:+a+cautionary+tale.">Using dendritic cell maturation and IL-12 producing capacity as markers of function: a cautionary tale.</a> J Immunother. 31, 359-369 (2008).</li><li>Leen, A. M., Myers, G. D., Sili, U., Huls, M. H., Weiss, H., Leung, K. S., Carrum, G., Krance, R. A., Chang, C. C., Molldrem, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoculture-derived+T+lymphocytes+specific+for+multiple+viruses+expand+and+produce+clinically+relevant+effects+in+immunocompromised+individuals.">Monoculture-derived T lymphocytes specific for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals.</a> Nat. Med. 12, 1160-1166 (2006).</li><li>Gerdemann, U., Christin, A. S., Vera, J. F., Ramos, C. A., Fujita, Y., Liu, H., Dilloo, D., Heslop, H. E., Brenner, M. K., Rooney, C. M., Leen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+of+DCs+to+generate+Multivirus-specific+T+cells+for+prevention+or+treatment+of+viral+infections+in+the+immunocompromised+host.">Nucleofection of DCs to generate Multivirus-specific T cells for prevention or treatment of viral infections in the immunocompromised host.</a> Mol. Ther. 17, 1616-1625 (2009).</li><li>Leen, A. M., Christin, A., Khalil, M., Weiss, H., Gee, A. P., Brenner, M. K., Heslop, H. E., Rooney, C. M., Bollard, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+hexon-specific+CD4+and+CD8+T-cell+epitopes+for+vaccine+and+immunotherapy.">Identification of hexon-specific CD4 and CD8 T-cell epitopes for vaccine and immunotherapy.</a> J Virol. 82, 546-554 (2008).</li><li>Leen, A. M., Christin, A., Myers, G. D., Liu, H., Cruz, C. R., Hanley, P. J., Kennedy-Nasser, A. A., Leung, K. S., Gee, A. P., Krance, R. A., Brenner, M. K., Heslop, H. E., Rooney, C. M., Bollard, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+T+lymphocyte+therapy+with+donor+T+cells+prevents+and+treats+adenovirus+and+Epstein-Barr+virus+infections+after+haploidentical+and+matched+unrelated+stem+cell+transplantation.">Cytotoxic T lymphocyte therapy with donor T cells prevents and treats adenovirus and Epstein-Barr virus infections after haploidentical and matched unrelated stem cell transplantation.</a> Blood. 114, 4283-4292 (2009).</li><li>Feuchtinger, T., Matthes-Martin, S., Richard, C., Lion, T., Fuhrer, M., Hamprecht, K., Handgretinger, R., Peters, C., Schuster, F. R., Beck, R., Schumm, M., Lotfi, R., Jahn, G., Lang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safe+adoptive+transfer+of+virus-specific+T-cell+immunity+for+the+treatment+of+systemic+adenovirus+infection+after+allogeneic+stem+cell+transplantation.">Safe adoptive transfer of virus-specific T-cell immunity for the treatment of systemic adenovirus infection after allogeneic stem cell transplantation.</a> Br J Haematol. 134, 64-76 (2006).</li><li>Bunde, T., Kirchner, A., Hoffmeister, B., Habedank, D., Hetzer, R., Cherepnev, G., Proesch, S., Reinke, P., Volk, H. D., Lehmkuhl, H., Kern, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+from+cytomegalovirus+after+transplantation+is+correlated+with+immediate+early+1-specific+CD8+T+cells.">Protection from cytomegalovirus after transplantation is correlated with immediate early 1-specific CD8 T cells.</a> J. Exp. Med. 201, 1031-1036 (2005).</li><li>Leen, A., Meij, P., Redchenko, I., Middeldorp, J., Bloemena, E., Rickinson, A., Blake, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+immunogenicity+of+Epstein-Barr+virus+latent-cycle+proteins+for+human+CD4(+)+T-helper+1+responses.">Differential immunogenicity of Epstein-Barr virus latent-cycle proteins for human CD4(+) T-helper 1 responses.</a> J Virol. 75, 8649-8659 (2001).</li><li>Bickham, K., Munz, C., Tsang, M. L., Larsson, M., Fonteneau, J. F., Bhardwaj, N., Steinman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EBNA1-specific+CD4++T+cells+in+healthy+carriers+of+Epstein-Barr+virus+are+primarily+Th1+in+function.">EBNA1-specific CD4+ T cells in healthy carriers of Epstein-Barr virus are primarily Th1 in function.</a> J. Clin. Invest. 107, 121-130 (2001).</li><li>Straathof, K. C., Leen, A. M., Buza, E. L., Taylor, G., Huls, M. H., Heslop, H. E., Rooney, C. M., Bollard, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+latent+membrane+protein+2+specificity+in+CTL+lines+from+patients+with+EBV-positive+nasopharyngeal+carcinoma+and+lymphoma.">Characterization of latent membrane protein 2 specificity in CTL lines from patients with EBV-positive nasopharyngeal carcinoma and lymphoma.</a> J. Immunol. 175, 4137-4147 (2005).</li><li>Hislop, A. D., Taylor, G. S., Sauce, D., Rickinson, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+responses+to+viral+infection+in+humans:+lessons+from+Epstein-Barr+virus.">Cellular responses to viral infection in humans: lessons from Epstein-Barr virus.</a> Annu. Rev. Immunol. 25, 587-617 (2007).</li><li>Steven, N. M., Annels, N. E., Kumar, A., Leese, A. M., Kurilla, M. G., Rickinson, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+early+and+early+lytic+cycle+proteins+are+frequent+targets+of+the+Epstein-Barr+virus-induced+cytotoxic+T+cell+response.">Immediate early and early lytic cycle proteins are frequent targets of the Epstein-Barr virus-induced cytotoxic T cell response.</a> J. Exp. Med. 185, 1605-1617 (1997).</li><li>Luke, J., Carnes, A. E., Hodgson, C. P., Williams, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+antibiotic-free+DNA+vaccine+vectors+utilizing+a+novel+RNA+based+plasmid+selection+system.">Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system.</a> Vaccine. 27, 6454-6459 (2009).</li><li>Williams, J. A., Luke, J., Johnson, L., Hodgson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pDNAVACCultra+vector+family:+high+throughput+intracellular+targeting+DNA+vaccine+plasmids.">pDNAVACCultra vector family: high throughput intracellular targeting DNA vaccine plasmids.</a> Vaccine. 24, 4671-4676 (2006).</li><li>Vera, J. F., Brenner, L. J., Gerdemann, U., Ngo, M. C., Sili, U., Liu, H., Wilson, J., Dotti, G., Heslop, H. E., Leen, A. M., Rooney, C. M. <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+pre-clinical+and+clinical+applications+using+Gas-permeable+Rapid+Expansion+cultureware+(G-Rex.">Accelerated production of antigen-specific T-cells for pre-clinical and clinical applications using Gas-permeable Rapid Expansion cultureware (G-Rex.</a> Journal of Immunotherapy. , (2009).</li><li>Vella, A. T., Dow, S., Potter, T. A., Kappler, J., Marrack, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine-induced+survival+of+activated+T+cells+in+vitro+and+in+vivo.">Cytokine-induced survival of activated T cells in vitro and in vivo.</a> Proc. Natl. Acad. Sci. U. S. A. 95, 3810-3815 (1998).</li><li>Vella, A., Teague, T. K., Ihle, J., Kappler, J., Marrack, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+4+(IL-4)+or+IL-7+prevents+the+death+of+resting+T+cells:+stat6+is+probably+not+required+for+the+effect+of+IL-4.">Interleukin 4 (IL-4) or IL-7 prevents the death of resting T cells: stat6 is probably not required for the effect of IL-4.</a> J. Exp. Med. 186, 325-330 (1997).</li><li>Heslop, H. E., Ng, C., Li, Y. C., Smith, C., A, C., Loftin, S. K., Krance, R. A., Brenner, M. K., Rooney, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+restoration+of+immunity+against+Epstein-Barr+virus+infection+by+adoptive+transfer+of+gene-modified+virus-specific+T+lymphocytes.">Long-term restoration of immunity against Epstein-Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes.</a> Nature Medicine. 2, 551-555 (1996).</li><li>Rooney, C. M., Smith, C. A., Ng, C., Loftin, S. K., Li, C., Krance, R. A., Brenner, M. K., Heslop, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+gene-modified+virus-specific+T+lymphocytes+to+control+Epstein-Barr+virus-related+lymphoproliferation.">Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr virus-related lymphoproliferation.</a> Lancet. 345, 9-13 (1995).</li><li>Einsele, H., Roosnek, E., Rufer, N., Sinzger, C., Riegler, S., Loffler, J., Grigoleit, U., Moris, A., Rammensee, H. G., Kanz, L., Kleihauer, A., Frank, F., Jahn, G., Hebart, 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, 3916-3922 (2002).</li><li>Walter, E. A., Greenberg, P. D., Gilbert, M. J., Finch, R. J., Watanabe, K. S., Thomas, E. D., Riddell, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+cellular+immunity+against+cytomegalovirus+in+recipients+of+allogeneic+bone+marrow+by+transfer+of+T-cell+clones+from+the+donor.">Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.</a> N. Engl. J. Med. 333, 1038-1044 (1995).</li></ol>

후안 F. 베라 윌슨 늑대 제조 컨설턴트입니다. 다른 저자는 관련 공개가 없습니다.

바이러스성 감염은 자신의 질병 또는 치료에 의해 immunocompromised 아르 환자에서 상당한 병적 상태와 사망률을 차지합니다. HSCT 후, 예를 들어, EBV와 CMV뿐만 아니라 호흡 Syncytial 바이러스 (RSV)와 같은 호흡기 바이러스와 같은 영구 herpesviruses에 의한 감염은 잘 알려진 반면 교수실, BK 바이러스에 의한 감염의 중요성, 인간 herpesvirus는 (HHV) -6 더 최근에는 감사하고 있습니다. 약리 대리인 어떤 감염에 대한 표준 치료하는 동안, 그들은 실질적인 toxicities을 가지고, 강한 변종을 생성하고, 자주 비효 있습니다. 대조적으로, 줄기 세포 기증자에서 파생된 바이러스 구체적인 T 세포는 hemopoietic 줄기 세포 이식에 바이러스 감염 또는 질병 (HSCT) 설정 2,5,6,18-21의 예방 및 치료에 안전하고 효과적인 입증했습니다. 그러나 T 세포 immunotherapy의 광범위한 구현 궁극적으로 CTL 생산에 필요한 비용, 복잡하고 시간에 따라 제한됩니다. 현재 원고에 기술된 multivirus CTL을 생성하기 위해 우리의 소설과 빠른 접근 방식은 실질적으로 immunocompromised 호스트에 대한 치료의 표준이 될 수있는 전략을 사용, 바이러스성 질환에 대한 세포 독성 T 세포 치료의 가능성을 증가한다. APCs로 플라스미드 nucleofected DC가의 사용은 다른 DC의 집단은 각각의 플라스미드 3 활용되기 때문에 하나의 셀 안에 표시되고 여러 바이러스 항원의 바이러스성 벡터는 물론 경쟁없이는 MHC 클래스 I 및 II 모두에서 항원 프레 젠 테이션을 수 있습니다. correspondingly 16,17 항원에 특정한 T 세포 응답의 주파수와 레퍼토리을 높일 수 있습니다 IL - 7분의 4 증가 T 세포 생존과 확산의 사용합니다. 마지막으로, G - 렉스의 문화는 크게 문화 중 T 세포 apoptosis을 줄일 수 있습니다. 가스 교환 (에서 O 2, CO 2 아웃)가 더 많은 영양소를 제공하고 폐기물을 diluting, 세포 위의 매체보다 깊이있게하면서 hypoxia을 방지, 술병의 바닥에 가스 투과 실리콘 막에 걸쳐 발생합니다. 이 플랫폼은 또한 보호 항원을 식별하는 경우 추가로 바이러스 확장될 수 있습니다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> CellGenix </td><td> CellGenix </td><td> 2005 </td></tr><tr><td> IL4 </td><td> R + D 시스템 </td><td> 204-IL/CF </td></tr><tr><td> IL7 </td><td> Peprotech </td><td> 200-15 </td></tr><tr><td> Hyclone RPMI 1640 </td><td> 써모 과학 </td><td> SH30096.01 </td></tr><tr><td> 인간 AB 혈청 </td><td> 밸리 바이오 메디컬 주식 회사 </td><td> HP1022 </td></tr><tr><td> Nucleofector </td><td> Amaxa / Lonza </td><td> AAF - 1001B &amp; AAF - 1001X </td></tr><tr><td> Nucleofection 키트 </td><td> Amaxa / Lonza </td><td> V4XP - 3012 </td></tr><tr><td> Plasmids </td><td> NTC </td><td> N / A </td></tr><tr><td> GM - CSF </td><td> R &amp; D </td><td> 215-GM/CF </td></tr><tr><td> IL1 </td><td> R &amp; D </td><td> 201 - LB - 025 </td></tr><tr><td> IL6 </td><td> R &amp; D </td><td> 206 - IL - CF </td></tr><tr><td> TNFα </td><td> R &amp; D </td><td> 210 - TA - 010 </td></tr><tr><td> PGE2 </td><td> 시그마 </td><td> P6532 - 1MG </td></tr><tr><td> G - 렉스 </td><td> 윌슨 울프 제조 </td><td> AY11 - 00027 </td></tr></tbody></table>

generation of multivirus-specific t cells to prevent/treat viral infections after allogeneic hematopoietic stem cell transplant

바이러스성 감염은 allogeneic 조혈 줄기 세포 이식 (HSCT) 수신자의 병적 상태와 사망률의 원인이됩니다. 우리와 다른 사람이 성공적으로 생성되고 엡스타인 바 바이러스 (EBV), 사이토 메갈로 바이러스 (CMV)와 monocytes와를 사용하여 아데노 바이러스 (교수실)에 대한 T - 세포를 특정 주입이 EBV - 변환 lymphoblastoid 셀 (EBV - LCL) 아데노 바이러스 벡터와 마찬가지로 유전자 - 수정 항원 제시 세포 (APCs). 마찬가지로 몇 가지 2x10으로 5 / kg trivirus 특정 세포 독성 T의 lymphocytes (CTL)은 주입 후 몇 가지 로그의 확산 및 다른 가능한 치료에 내성도 심각한 바이러스성 질병을 예방하고 치료 나타났다. 준비 -이 장려 접근법의 광범위한 구현은 높은 생산 비용, 제조의 복잡 성과 연장 시간 (총 10-14주 CTL 제조 EBV - LCL의 생성 및 4~8주에 대한 4~6주)에 의해 제한됩니다. 이러한 한계를 극복하기 위해 우리는 새로운 GMP 준수 CTL 생산 프로토콜을 개발했습니다. 첫째, 우리는 항원 제시와 같은 DNA plasmids 인코딩 LMP2, EBNA1 및 BZLF1 (EBV), Hexon 및 펜톤 (교수실), 그리고 pp65 및 IE1 (CMV)와 nucleofected 돌기 세포 (DCS)를 사용하여 T - 세포를 자극하는 adenovectors 대신에 세포. 이러한 APCs는 모든 자극 항원에 대한 T 세포가 특정 재활 성화. 둘째, 문화는 IL - 4의 존재에 T - 세포를 활성화 (1,000 U / ML)와 IL - 7 (10ng/ml)은 증가 및 라인에서 특정 T 세포의 레퍼토리와 주파수를 부양하고 있습니다. 셋째, 우리는 따라서 EBV - LCLs에 대한 요구 사항을 제거하고 기술자의 개입을 줄이고, 하나의 자극 후 대형 세포 숫자의 확장과 생존을 촉진 새로운 가스 투과 문화 장치를 (G - 렉스) 사용하고 있습니다. 이러한 변경 사항을 구현함으로써 우리는 지금&gt; 90%에 의해 줄어 듭니다 10 6 세포 당 비용 EBV, CMV 및 교수실을 대상으로 multispecific CTL를 생산하고, 수있는 추가로 연장할 수 있습니다 접근법을 사용 단 십일 오히려 10 주 보호 바이러스 항원. 우리의 FDA 승인 접근 방식은 높은 위험 allogeneic HSCT받는 사람에 대한 예방 및 치료 응용 프로그램에 가치가 있어야합니다.

Watch this Scientific Journal Video about Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant at JoVE.com

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

의 생성에 대한 신속, 간단하고 비용 효율적인 프로토콜 기증자 파생 CMV, EBV 감염 또는 교수실을 개발하는 위험 allogeneic 조혈 줄기 세포 이식 (HSCT) 수신자에게 주입을위한 multivirus 특정 CTLs (rCTL을). 이 제조 과정은 GMP 준수하고 전문 센터 이외 T - 세포 immunotherapy의 광범위한 구현을 보장한다.

Multivirus 특정 T 세포 생성 Allogeneic 조혈 줄기 세포 이식 후 바이러스 감염을 예방 / 치료

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

generation-multivirus-specific-t-cells-to-preventtreat-viral

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

17.1K 조회수.  Baylor College of Medicine. 의 생성에 대한 신속, 간단하고 비용 효율적인 프로토콜 기증자 파생 CMV, EBV 감염 또는 교수실을 개발하는 위험 allogeneic 조혈 줄기 세포 이식 (HSCT) 수신자에게 주입을위한 multivirus 특정 CTLs (rCTL을). 이 제조 과정은 GMP 준수하고 전문 센터 이외 T - 세포 immunotherapy의 광범위한 구현을 보장한다.

비디오: Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

마우스 T - 세포에서 T - 세포 수용체의 Retroviral 전달

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

연구

면역학 및 감염병학

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

에 셀 운명을 따라<em&gt; E. 대장균</em&gt; 파지 람다에 의해 감염 후

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

기본 인적 나이브 및 메모리 T의 세포로부터 유도된 규제 T 세포의 생성

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

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 ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

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

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

정제 된 조혈 줄기 / 전구 세포에 DNA 돌연변이를 확인

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

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

마우스에 동종 CD4 T 세포 반응을 연구하기 위해 테일 피부 이식

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

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

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

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

사이토 카인 캡처 시스템을 사용하여 임상 응용 프로그램에 대한 거대 세포 바이러스 고유의 자동화 된 세포 농축은 T 세포

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

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

세대와의 멀티 표현형 높은 콘텐츠 상영<em&gt; Coxiella burnetii의</em&gt; 트랜스포존 돌연변이

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

자가 면역 대해 줄기 세포 유래의 항원 특이 적 조절 T 세포의 발달

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

이미징 유동 세포 계측법을 사용하여 T 세포 Alloreactivity의 측정

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...