이 작품은 치료 재단과 NIH / NCI 9R01 CA152045 및 P50-CA116201를위한 수잔 G. 코멘에서 키스 L. Knutson의에 교부금에 의해 지원되었다.

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	<li>Zhang, N., Bevan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8(+)+T+cells:+foot+soldiers+of+the+immune+system.">CD8(+) T cells: foot soldiers of the immune system.</a> Immunity. 35, 161-168 (2011).</li><li>Garboczi, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+complex+between+human+T-cell+receptor,+viral+peptide+and+HLA-A2.">Structure of the complex between human T-cell receptor, viral peptide and HLA-A2.</a> Nature. 384, 134-141 (1996).</li><li>Knutson, K. L., dela Rosa, C., Disis, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+analysis+of+T-cell+immunity.">Laboratory analysis of T-cell immunity.</a> Front Biosci. 11, 1932-1944 (2006).</li><li>Andersen, M. H., Schrama, D., Thor Straten, P., Becker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cytotoxic T cells. J. Invest. Dermatol. 126, 32-41 (2006).</li><li>Weren, A., Bonnekoh, B., Schraven, B., Gollnick, H., Ambach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+flow+cytometric+assay+focusing+on+perforin+release+mechanisms+of+cytotoxic+T+lymphocytes.">A novel flow cytometric assay focusing on perforin release mechanisms of cytotoxic T lymphocytes.</a> J. Immunol. Methods. 289, 17-26 (2004).</li><li>Aktas, E., Kucuksezer, U. C., Bilgic, S., Erten, G., Deniz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+CD107a+expression+and+cytotoxic+activity.">Relationship between CD107a expression and cytotoxic activity.</a> Cell Immunol. 254, 149-154 (2009).</li><li>Knutson, K. L., Schiffman, K., Disis, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+with+a+HER-2/neu+helper+peptide+vaccine+generates+HER-+2/neu+CD8+T-cell+immunity+in+cancer+patients.">Immunization with a HER-2/neu helper peptide vaccine generates HER- 2/neu CD8 T-cell immunity in cancer patients.</a> J. Clin. Invest. 107, 477-484 (2001).</li><li>Asiedu, M. K., Ingle, J. N., Behrens, M. D., Radisky, D. C., Knutson, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta/TNF(alpha)-mediated+epithelial-mesenchymal+transition+generates+breast+cancer+stem+cells+with+a+claudin-low+phenotype.">TGFbeta/TNF(alpha)-mediated epithelial-mesenchymal transition generates breast cancer stem cells with a claudin-low phenotype.</a> Cancer Res. 71, 4707-4719 (2011).</li><li>Strijbosch, L. W., Buurman, W. A., Does, R. J., Zinken, P. H., Groenewegen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+dilution+assays.+Experimental+design+and+statistical+analysis.">Limiting dilution assays. Experimental design and statistical analysis.</a> J. Immunol. Methods. 97, 133-140 (1987).</li><li>Gallimore, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+exhaustion+of+lymphocytic+choriomeningitis+virus-specific+cytotoxic+T+lymphocytes+visualized+using+soluble+tetrameric+major+histocompatibility+complex+class+I-peptide+complexes.">Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes.</a> J. Exp. Med. 187, 1383-1393 (1998).</li></ol>

생산이 비디오 문서의 무료 액세스가 로슈 진단에 의해 후원됩니다.

CD8 T 세포에 직접 감염된 또는 악성 세포에게 4를 죽일 수있는, 적응 면역 반응에 중요하다. 항원에 특정한 CD8 T 세포 (예 : ELISPOT, 사량, 유동 세포 계측법)의 수를 측정하는 방법은 여러 가지가 있지만, 종종 이러한 세포가 3 기능적 세포 독성 여부를 알고 도움이됩니다. 세포 독성 기능을 평가하기위한 가장 인식 할 수있는 분석은 황금 표준 분석 4 간주됩니다 CRA입니다. CRA의 주요 한계는 방사능 (1) 요구 사항, 감도 (2) 부족, 기계론의 정보 (3) 부족, 세포의 대량 (4)를위한 요구 사항, 그리고 (5) 힘드는 세트까지입니다. 따라서, 지난 10 년 동안, 이러한 한계를 최소화하기 위해 새로운 전략을 채택에 관심이있어왔다. 새로운 접근 방식을 포함하는 측정하는 caspase의 릴리스 4, BLT 에스 테라 activity5, 및 CD107 6 표면 발현.에게 본 논문은 제안하는로슈 xCelligence 시스템에서 수행 임피던스 기반의 분석은 또한 CRA에 대한 대안으로 유용 할 수 있습니다. 임피던스 기반의 접근 방식을 적용 할 수있는 능력은 표면에 단단히 부착하지 않는 T 세포, 따라서 측정과 T 세포의 직접적인 간섭의 우려를 감소 임피던스에 많은 영향을 미칠 것으로 나타나지 않는 것을 관찰에 달려있다. 임피던스 분석 시스템 사용에 필요한 노동력은 훨씬 적은 주로 방사능의 사용에 대한 규제 문제의 제거로 인해 것으로 예상된다. 임피던스 기반 시스템의 유일한 주목할만한 단점은 50,000 달러에서 $ 60,000 지출이 xCelligence 단위의 구입이 필요합니다 것입니다. 그러나 xCelligence 장치는 방사선 카운터에 대한 필요성을 제거하고 T 세포 독성 8 이외에 약물 및 마이그레이션에 대한 세포 반응을 평가하는데 유용되고, 훨씬 더 다재다능하다. 그 관찰종양 세포의 임피던스 감소가 용량 의존적이며, 같은 간단한 보조 차 함수에 따라 합리적으로 가파른 기울기, 그림 2는 분석이 9 세포 독성 세포의 빈도를 추정하기 위해 희석 분석 (LDAs)를 제한하는 데 사용할 수 있다고 제안 . 임피던스 기반의 접근 방식을 가진 하나의 장점은 필요한 T 세포와 종양 세포의 수가 LDA는 잠재적으로 더 가능하고, CRA 미만이다. 예를 들어, CRA에 1시 40분 비율로 잘 당 1 × 10 5 종양 세포뿐만 당 4 X 10 6 총 T 세포를 필요로하는 도금입니다. 그러나, 임피던스 기반의 분석에 잘 당 1.5 × 10 4 종양 세포가 잘 당 6 × 10 5 총 T 세포를 소모하는 필요합니다. 따라서, 임피던스 기반의 분석은 85 % 이하 T 세포와 종양 세포를 사용합니다. 이 감소는 임피던스 기반의 시스템은 인간의 임상 실험에서 면역 감시에 적용 할 수 있다고 제안 여기서 t혈액의 그 볼륨은 제한 될 수 있습니다립니다. CRA가 하나의 시점에 제한되는 동안 또한, 임피던스 기반의 분석은 사용자가 최소한의 추가 노동과 세포와 같은 실험을 여러 용해 시점을 비교할 수 있도록, 지속적인 실시간 데이터를 수집합니다. 주파수 계산은 잠재적으로 총 10 항원에 특정한 CD8 T 세포에 기능적인 세포 독성 T 세포의 비율을 설정하는 등 사량 분석 등의 기능을 측정하지 않는 다른 전략과 결합 될 수있다. 임피던스 기반의 접근 방식의 가장 매력적인 기능 중 하나는 다른 대부분의 분석과는 달리, 세포 독성이 잠재적으로 쉽게 억제제 또는 항체의 사용과 세포의 상호 작용을 연구하고, 실시간으로 감시 할 수 있다는 것입니다. 또 다른 방법은 라벨에 저항 종양 세포에 적합 할 수 있다는 것입니다. 비 살해 종양 세포를 표시하고 유지되지 않기 때문에 마지막으로, 살균, 더 수확 T 세포 내성 세포 연구 possib입니다르. 두 시스템이 특정 T 세포의 질문에 답변 종양 살해 데이터를 제공하지만 요약하면, 임피던스 기반의 분석은 CRA보다 더 민감하고 더 유연한 플랫폼을 제공합니다.

<table><tbody><tr><td>Ficoll-Paque Plus </td><td>GE Healthcare</td><td>17-1440-03</td><td>Used to isolate PBMCs from blood </td></tr><tr><td>RPMI 1640 </td><td>Cellgro</td><td>10-040-CV</td><td>Cell culture media </td></tr><tr><td>FBS </td><td>Denville Scientific</td><td>FB5001-H</td><td>Cell culture media </td></tr><tr><td>Human IL-2 </td><td>eBioscience</td><td>14-8029-81</td><td>For stimulating growth of T cells </td></tr><tr><td>Human Interferon gamma </td><td>Cell Sciences</td><td>CR2026</td><td>For stimulating MHC expression in tumor cells </td></tr><tr><td>HER-2/neu p369-377 peptide </td><td>Mayo Clinic peptide synthesis core</td><td>amino acid sequence KIFGSLAFL</td><td>Binds to HLA-A2 </td></tr><tr><td>Influenza p58-66 peptide </td><td>Mayo Clinic peptide synthesis core</td><td>amino acid sequence GILGFVFTL</td><td>Binds to HLA-A2 </td></tr><tr><td>Human CD3/CD28 Dynabeads </td><td>Invitrogen</td><td>111-61D</td><td>For non-specific T cell activation and expansion </td></tr><tr><td>Chromium-51 </td><td>MP Biomedicals</td><td>62015</td><td>CRA labeling reagent </td></tr><tr><td>E-plate </td><td><a href="http://www.aceabio.com/" target="_blank"><img src="/img/new_window.png" /> Roche Group</a></td><td>05469830001</td><td>For xCelligence impedance unit </td></tr><tr><td>Luma plate 96 </td><td>PerkinElmer, Inc.</td><td>6005630</td><td>For measuring radioactivity in the Top Count NXT. </td></tr><tr><td>xCelligence RTCA DP Station </td><td><a href="http://www.aceabio.com/" target="_blank"><img src="/img/new_window.png" /> Roche Applied Science</a></td><td>RTCA DP</td><td>Impedance unit </td></tr><tr><td>Top Count NXT </td><td>PerkinElmer, Inc.</td><td>C990601</td><td>Scintillation &amp;amp; Luminescence Counter </td></tr><tr><td>Fas Ligand antibody</td><td>BD Biosciences</td><td>556372</td><td>For blocking Fas-Fas ligand interactions</td></tr><tr><td>HLA-A2 BB7.2 antibody</td><td>BD Biosciences</td><td>551230</td><td>For blocking HLA class I</td></tr><tr><td>Mouse IgG2b, &amp;#954;</td><td>BD Biosciences</td><td>555740</td><td>Isotype control for HLA class I</td></tr><tr><td>SKBR3 tumor line</td><td>ATCC</td><td>HTB-30</td><td>Adenocarcinoma</td></tr><tr><td>MCF7 tumor line</td><td>ATCC</td><td>HTB-22</td><td>Adenocarcinoma</td></tr><tr><td>HCC1419 tumor line</td><td>ATCC</td><td>CRL-2326</td><td>Primary ductal carcinoma</td></tr><tr><td>BT20 tumor line</td><td>ATCC</td><td>HTB-19</td><td>Carcinoma</td></tr></tbody></table>

determining optimal cytotoxic activity of human her2neu specific cd8 t cells by comparing the cr51 release assay to the xcelligence system

세포 독성 CD8 T 세포가 감염되거나 악성 숙주 세포 1의 죽음을 유도 할 수있다 T 세포의 하위 그룹을 구성합니다. 이러한 세포는 특수한 수용체를 내가 2 분자가 HLA 클래스에 바인드 특정 항원 펩티드를 인식 할 수있는 T 세포 수용체 (TCR)라고 표현한다. 이러한 목표 세포의 죽음의 결과로 granzymes와 퍼포 같은 용해성 물질의 생산에 자신의 HLA 클래스 I 분자 결과를 통해 감염된 세포 나 종양 세포의 참여. 이 같은 ELISPOT 또는 흐름 세포 계측법으로 분석을 사용하여 항원 특이 CD8 T 세포의 주파수를 결정하는 데 유용하지만, 그것은 세포 독성 분석에게 3를 사용하여 CD8 T 세포 반응의 강도를 확인하기 위해 도움이됩니다. 세포 독성 기능을 평가하기위한 가장 인식 할 수있는 분석은 표준 분석 4 간주되는 크롬 출시 분석 (CRA)입니다. CRA는 감마 방사선에 세포의 노출, L 등 여러 가지 한계를 가지고재현성 및 세포의 큰 숫자에 대한 요구 사항의 ACK. 지난 10 년간 이러한 한계를 극복하기 위해 새로운 전략을 채택에 관심이있어왔다. 새로운 접근 방식을 포함하는 측정하는 caspase의 릴리스 4, BLT의 에스테르 가수 분해 효소 활동 5와 CD107 6 표면 발현.에게 로슈 xCelligence 시스템을 사용하여 임피던스 기반의 분석은, CRA에 대한 대안으로 가능성을 위해 본 논문에서 조사 하였다. 자기편 종양 세포가 전극 판에 바인딩 할 때 임피던스 또는 전류에 대한 반대가 발생합니다. 종양 세포 살해 후 분리 및 전기 임피던스는 xCelligence 시스템에 의해 측정 할 수있는 줄어 듭니다. 세포 매개 살인을 평가하기 위해 임피던스 기반의 접근 방식을 적용 할 수있는 능력은 T 세포는 대부분의 표면에 단단히 부착하지 않고 따라서 T 세포의 직접적인 간섭의 우려를 감소 임피던스에 많은 영향을 미칠 것으로 나타나지 않는 것을 관찰에 달려있다 와측정. 결과는 임피던스 기반의 분석은 표준 CRA에 상대적으로 증가 감도 항원 특이 세포 독성 CD8 T 세포의 수준의 변화를 감지 할 수 보여줍니다. 이러한 결과를 바탕으로, 임피던스 기반의 접근 방식은 CRA의 또는 세포 독성 CD8 T 세포의 기능을 측정하는 것을 목표로 다른 방법에 대한 좋은 대안이 될 수 있습니다.

Watch this Scientific Journal Video about Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System at JoVE.com

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

크롬 출시 분석, 세포 독성 T 세포의 활동을 검출하기위한 일반적인 분석은 몇 가지 제한 사항이 있습니다. 본 문서에서, 항원 특이 CD8 T 세포와 인간의 유방암 종양 라인 SKBR3를 사용하여 임피던스 기반의 접근 방식은 세포의 살해를 감지하는 기능에 대해 조사 하였다.

xCELLigence 시스템으로 CR51 릴리스 분석을 비교하여 인간 Her2neu 특정한 CD8 T 세포의 최적 세포 독성 활동 결정

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

determining-optimal-cytotoxic-activity-human-her2neu-specific-cd8-t

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

24.9K 조회수.  College of Medicine, Mayo Clinic. 크롬 출시 분석, 세포 독성 T 세포의 활동을 검출하기위한 일반적인 분석은 몇 가지 제한 사항이 있습니다. 본 문서에서, 항원 특이 CD8 T 세포와 인간의 유방암 종양 라인 SKBR3를 사용하여 임피던스 기반의 접근 방식은 세포의 살해를 감지하는 기능에 대해 조사 하였다.

비디오: Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

체 외 종양 세포 Rechallenge 공상 항 원 수용 체 T 세포 Antitumor 기능 예측 평가

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

연구

암 연구학

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

T 세포 양적 높은 처리량 단일 세포 세포 독성 분석

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

면역학 및 감염병학

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

항체 의존성 세포 독성 변형 화합물의 식별을 위한 High-content screening assay

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

A Nonviral Approach to Generate Transient Chimeric Antigen Receptor T Cells Using mRNA for Cancer Immunotherapy

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain hematological malignancies such as lymphomas and leukemias. However, as more cancer patients receive CAR-T cell therapies, treatment-associated secondary primary malignancies are increasingly being reported partly due to unexpected CAR transgene insertion, raising serious safety concerns. To address this issue, we describe here a nonviral, non-integrating approach to generate transient CAR-T cells using mRNA. We electroporated T cells with modified mRNA encoding a human epidermal growth factor receptor 2 (HER2)-specific CAR and generated transient HER2-targeted CAR-T cells. The CAR was efficiently expressed on the T cell surface 1 day after electroporation, increased by day 2, and dramatically declined by day 5. The transient CAR-T cells exhibited potent cytotoxicity against HER2-positive SKOV-3 ovarian cancer cells and secreted high levels of IFN-&#978;. This protocol provides a step-by-step guide for developing small-scale transient CAR-T cells without permanent CAR transgene integration, describing detailed procedures for preparation of CAR mRNA, activation and transfection of T cells, assessment of CAR expression, and in vitro analysis of CAR-T cell function. This method is suitable for transient CAR-T cell generation in both preclinical and clinical studies.

This protocol outlines a detailed procedure for generating transient chimeric antigen receptor (CAR) T cells using non-integrating mRNA for cancer immunotherapy and provides reliable methods for evaluating CAR-T cells and their cytotoxic function.

암 면역 요법을 위해 mRNA를 사용하여 일시적인 키메라 항원 수용체 T 세포를 생성하는 비바이러스 접근법

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain ...

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and activity of cytotoxic T cells within tumors and their tumor microenvironment (TME) are now well-established prognostic and predictive biomarkers for numerous tumor types. However, it is well-known that various tumor treatment modalities, including radiotherapy, chemotherapy, immunotherapy, and targeted therapy, modulate not only the immunogenicity of the tumor but also the immune system itself. Consequently, the interaction between tumor cells and T cells requires more intensive study in different therapeutic contexts to fully understand the complex role of T cells during tumor therapy. To address this need, a protocol was developed to analyze the activity and proliferative capacity of human cytotoxic (CD8+) T cells in co-culture with pre-treated tumor cells. Specifically, CD8+ T cells from healthy donors are stained with the non-toxic proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated using CD3/CD28-coated plates. Subsequently, T cells are co-cultured with pre-treated tumor cells. As a readout, T cell proliferation is quantified by measuring CFSE signal distribution and assessing the expression of surface activation markers via flow cytometry. This can be further complemented by quantifying cytokine release using enzyme-linked immunosorbent assay (ELISA). This method facilitates the evaluation of treatment-induced changes in the interaction between tumor cells and T cells, providing a foundation for more detailed analyses of tumor treatment modalities and their immunogenicity in a human ex vivo setting. Additionally, it contributes to the reduction of pre-clinical in vivo analyses.

The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

전처리된 종양 세포의 동종 공동 배양 환경에서의 인간 T 세포 활성 분석

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

고체 및 혈액암 세포를 표적으로 하는 키메라 항원 수용체 T 세포를 위한 실시간 힘 분석

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

종양 특이 CD8+ T 세포 반응에서 면역 우세를 연구 하기 위한 생체 내 세포 독성 분석 법

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

<meta charset="utf8"></head><body>암 면역 요법을 위한 ADCC 생물학적 검정에서 효과기로서의 갑상선 암 세포

Assessing Antibody-dependent, Cell-mediated Cytotoxicity in Cancer Cells using Antibody-Dependent Cell-Mediated Cytotoxicity Reporter Bioassay

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in cancer immunotherapy. Evaluating ADCC activity in cancer cells is essential for the development and optimization of antibody-based treatments. Here, we propose a methodological approach of utilizing an ADCC bioassay kit for quantitative assessment of ADCC reaction using thyroid cancer cells as effector cells. The protocol involves the co-culture of effector cells with target cancer cells in different ratios in the presence of a therapeutic antibody. The ADCC bioassay kit used in this experiment includes the genetically engineered effector cells expressing a luciferase reporter gene under the control of Nuclear Factor of Activated T-cell (NFAT) response elements. Upon the binding of the surface antigen on the target cells with the antibodies and effector cells, the effector cells release luciferase, enabling quantification of cytotoxicity through measurement of luminescence signal. In contrast to conventional ADCC assays, this method proved the binding of target antigen with antibodies and effector cells, which can produce reliable results in a short period.

<meta charset="utf8"></head><body>저자 스포트라이트: 항체 의존성, 세포 매개 세포독성 분석을 통한 항체 기반 암 치료 최적화

Here, we present a protocol for an antibody-dependent, cell-mediated cytotoxicity (ADCC) assay using an ADCC bioassay kit. This method offers a valuable tool for elucidating the ADCC mechanism and evaluating the therapeutic potential of antibodies in cancer immunotherapy.

항체 의존성 세포 매개 세포 독성 리포터 생물학적 분석을 사용하여 암세포의 항체 의존성, 세포 매개 세포 독성 평가

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in ...

xCELLigence 시스템으로 CR51 릴리스 분석을 비교하여 인간 Her2neu 특정한 CD8 T 세포의 최적 세포 독성 활동 결정

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체 외 종양 세포 Rechallenge 공상 항 원 수용 체 T 세포 Antitumor 기능 예측 평가

T 세포 양적 높은 처리량 단일 세포 세포 독성 분석

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

항체 의존성 세포 독성 변형 화합물의 식별을 위한 High-content screening assay

암 면역 요법을 위해 mRNA를 사용하여 일시적인 키메라 항원 수용체 T 세포를 생성하는 비바이러스 접근법

전처리된 종양 세포의 동종 공동 배양 환경에서의 인간 T 세포 활성 분석

고체 및 혈액암 세포를 표적으로 하는 키메라 항원 수용체 T 세포를 위한 실시간 힘 분석

종양 특이 CD8⁺ T 세포 반응에서 면역 우세를 연구 하기 위한 생체 내 세포 독성 분석 법

항체 의존성 세포 매개 세포 독성 리포터 생물학적 분석을 사용하여 암세포의 항체 의존성, 세포 매개 세포 독성 평가

항체 의존성 세포 매개 세포 독성 리포터 생물학적 분석을 사용하여 암세포의 항체 의존성, 세포 매개 세포 독성 평가