この作品は、キュア財団とNIH / NCI 9R01 CA152045とP50-CA116201ためのスーザンG.コーメンからキースL.ナットソンへの補助金によって支えられている。

<ol>
	<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>

生産とこのビデオ記事の無料アクセスは、ロシュ·ダイアグノスティックス社が主催しています。

CD8 T細胞が直接4感染または悪性細胞を殺すことができるという、適応免疫応答に重要です。抗原特異的CD8 T細胞（例えばELISPOT、テトラマー、フローサイトメトリー）の数を測定するいくつかの方法があるが、多くの場合、それは、これらの細胞が機能的に3細胞毒性であるかどうかを知ることは有用である。細胞傷害性機能を評価するための最も有名なアッセイは、ゴールドスタンダードアッセイ4みなされるCRA、です。 CRAの主な制限は、放射能の（1）の要件、感度、（2）不足、機械的な情報（3）不足している、細胞の多量のために（4）の要件、および（5）面倒なセットアップです。従って、過去十年間、これらの制限を最小限に抑えるために新たな戦略を採用するに関心があった。新しいアプローチは、それらのそのメジャーカスパーゼリリース4、BLTエステラーゼactivity5、表面CD107 6の発現を含む。現在の原稿は示唆しているロシュxCELLigenceシステム上で実行インピーダンスベースのアッセイも、CRAの代替として有用であり得る。インピーダンスベースのアプローチを適応する能力は、表面にしっかりと付着しないT細胞は、測定したT細胞の直接干渉の任意の関心を減少させるため、インピーダンスに大きな影響を持っているようには見えないという観察に基づいている。インピーダンス測定システムを使用するために必要な労力を大幅に減らす放射能の使用のための規制懸念の除去が主な原因であると予想される。インピーダンスベースのシステムの唯一の顕著な欠点は5万ドルから$ 60,000支出がエクセリジェンスユニットの購入のために必要とされることである。しかし、エクセリジェンス部は、放射線カウンタの必要性を排除し、T細胞の細胞毒性8に加えて、薬物および移行の細胞応答を評価するために有用である、はるかに汎用性があります。 その観察単純な二次関数は、次合理的に急勾配のアッセイが細胞傷害性細胞9の周波数を推定するために、限界希釈アッセイ（のLDA）で使用することができることを示唆して腫瘍細胞インピーダンスの減少は、 図2に示すように、用量依存性である。インピーダンスに基づくアプローチの一つの利点は、必要なT細胞および腫瘍細胞の数は、LDAが潜在的により実現すること、CRA未満であることである。例えば、CRAで、午前1時40分の割合で、ウェル当たり1×10 5腫瘍細胞はウェルあたり4×10 6合計T細胞を必要とするメッキです。しかし、インピーダンスベースのアッセイで、ウェルあたりわずか1.5×10 4個の腫瘍細胞がウェルあたりわずか6×10 5総T細胞を消費する、必要とされている。従って、インピーダンスに基づくアッセイは、85％より少ないT細胞および腫瘍細胞を使用する。この減少は、インピーダンスに基づくシステムは、ここで、tヒト臨床試験において免疫学的監視に適合させることができることを示唆している血描くの彼のボリュームが制限される場合があります。 CRAは、単一の時点に限定されている間も、インピーダンスに基づくアッセイは、ユーザは、最小限の追加労力と細胞と同じ実験で複数の溶解時間点を比較することができ、連続的なリアルタイムデータを収集する。周波数の計算は、潜在的に全体の抗原特異的CD8 T細胞10に機能的な細胞傷害性T細胞の比を確立するテトラマー解析などの機能を測定していない他の戦略と組み合わせることができる。インピーダンスベースのアプローチの最も魅力的な特徴の一つは、他のほとんどのアッセイとは異なり、細胞毒性が潜在的にそれが簡単阻害剤または抗体の使用による細胞の相互作用を研究すること、リアルタイムでモニターすることができる、ということである。別の方法は、ラベリングレジスト腫瘍細胞に適していることである。最後に、非殺さ腫瘍細胞を標識し、滅菌、さらに収穫のままとT細胞耐性細胞の研究はpossibているされていないので、ル。 両方のシステムが特定のT細胞に反応して腫瘍を殺すのデータを提供しながら要約すると、インピーダンスベースのアッセイは、CRAよりも敏感で、より柔軟なプラットフォームを提供します。

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments</td>
 </tr>
 <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>Roche Applied Science </td>
 <td>05469830001 </td>
 <td>For xCelligence impedance unit </td>
 </tr>
 <tr>
 <td>Luma plate 96 </td>
 <td>Perkin Elmer </td>
 <td>6005630 </td>
 <td>For measuring radioactivity in the Top Count NXT. </td>
 </tr>
 <tr>
 <td>xCelligence RTCA DP Station </td>
 <td>Roche Applied Science </td>
 <td>RTCA DP </td>
 <td>Impedance unit </td>
 </tr>
 <tr>
 <td>Top Count NXT </td>
 <td>Perkin Elmer </td>
 <td>C990601 </td>
 <td>Scintillation &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, &kappa;</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

細胞傷害性CD8 T細胞は、感染又は悪性宿主細胞1の死を誘導することができるT細胞のサブグループを構成している。これらの細胞は、I 2分子の HLAクラスに結合した特定の抗原性ペプチドを認識することができ、T細胞受容体（TCR）と呼ばれる特殊な受容体を発現する。例えば、標的細胞死をもたらすグランザイムとパーフォリンとして溶解分子の産生におけるそれらのHLAクラスI分子結果介して感染細胞または腫瘍細胞の係。そのようなELISPOTまたはフローサイトメトリーなどのアッセイを用いて抗原特異的CD8 T細胞の頻度を決定することは有用であるが、それは細胞毒性アッセイ3を用いてCD8 T細胞応答の強度を確認するためにも有用である。細胞傷害性機能を評価するための最も有名なアッセイは、標準アッセイ4みなされるクロム放出アッセイ（CRA）、です。 CRAは、ガンマ放射線、lの細胞の曝露を含むいくつかの制限を有している再現性の確認応答、および多数の細胞のための要件。過去10年間で、これらの制限を克服するための新たな戦略を採用することに関心があった。新しいアプローチは、それらのそのメジャーカスパーゼリリース4、BLTのエステラーゼ活性5およびCD107 6の表面発現を含む。ロシュxCELLigenceシステムを用いたインピーダンスベースのアッセイは、CRAの代替としての可能性のために本論文​​で検討した。付着腫瘍細胞が電極板に結合したときの電流に対するインピーダンスまたは反対が発生します。腫瘍細胞は死滅し、次の電気インピーダンスを切り離しxCELLigenceシステムによって測定することができる低減される。細胞媒介性死滅を評価するために、インピーダンスベースのアプローチを適応する能力は、T細胞は、ほとんどの表面に強固に付着せず、従って、T細胞の直接干渉の任意の懸念を減少インピーダンスに大きな影響を持っているようには見えないという観察に基づいているととも​​に測定。結果は、インピーダンスに基づくアッセイは、標準CRAに対して増加した感度で抗原特異的細胞傷害性CD8 T細胞のレベルの変化を検出することができることを示している。これらの結果に基づいて、インピーダンスベースのアプローチは、細胞傷害性CD8 T細胞の機能を測定することを目的とCRAの又は他のアプローチへの良好な代替物とすることができる。

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

クロム放出アッセイ、細胞傷害性T細胞活性を検出するための一般的なアッセイは、いくつかの制限を有する。抗原特異的CD8 T細胞およびヒト乳癌腫瘍ライン、SKBR3を用いて、本資料では、インピーダンスベースのアプローチは、細胞死滅を検出する能力を調べた。

xCELLigenceシステムにCR51リリースアッセイを比較することによって、人間Her2neu特異的CD8 T細胞の最適な細胞傷害活性を決定する

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

Research

JoVE Journal

Immunology and Infection

24.9K ビュー.  College of Medicine, Mayo Clinic. クロム放出アッセイ、細胞傷害性T細胞活性を検出するための一般的なアッセイは、いくつかの制限を有する。抗原特異的CD8 T細胞およびヒト乳癌腫瘍ライン、SKBR3を用いて、本資料では、インピーダンスベースのアプローチは、細胞死滅を検出する能力を調べた。

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

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.

傷害性Tリンパ球によって媒介ベータ細胞死を評価するための方法

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

免疫・感染学

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

インビトロアッセイで HIV特異的CD4 T細胞エフェクター機能に対する免疫調節経路の影響を評価する

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

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 ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1感染CD4の生得センシングを評価<sup&gt; +</sup&gt;使用して形質細胞様樹状細胞によるT細胞<em&gt;エクスビボ</em&gt;共培養系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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.

末梢血からのヒトアロ抗原特異的T細胞の生成

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

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.

サイトカインキャプチャシステムを用いた臨床応用のためのサイトメガロウイルス特異的T細胞の自動化された細胞濃縮

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

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.

自己免疫に対する幹細胞由来の抗原特異的制御性T細胞の開発

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

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

サル免疫不全ウイルス特異的CD8の分析<sup&gt; +</sup&gt;ペプチドMHC-I四量体染色によりアカゲザルにおけるT細胞

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled &#34;Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.&#34; The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

その場でMHC テトラマー染色と場所、豊富と組織における抗原特異的 cd8 陽性 T 細胞の表現型を決定するための定量解析

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

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.

腫瘍特異的 CD8+ T 細胞応答における Immunodominance を研究するためのインビボ細胞毒性アッセイ

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