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	<li>Ruscetti, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+effector+mechanisms+against+HTLV-I-+and+HTLV-III/LAV-infected+lymphoid+cells.">Analysis of effector mechanisms against HTLV-I- and HTLV-III/LAV-infected lymphoid cells.</a> J Immunol. 136, 3619-3624 (1986).</li><li>Zheng, Z. Y., Zucker-Franklin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apparent+ineffectiveness+of+natural+killer+cells+vis-a-vis+retrovirus-infected+targets.">Apparent ineffectiveness of natural killer cells vis-a-vis retrovirus-infected targets.</a> J Immunol. 148, 3679-3685 (1992).</li><li>Ferrari, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clade+B-based+HIV-1+vaccines+elicit+cross-clade+cytotoxic+T+lymphocyte+reactivities+in+uninfected+volunteers.">Clade B-based HIV-1 vaccines elicit cross-clade cytotoxic T lymphocyte reactivities in uninfected volunteers.</a> Proc Natl Acad Sci U S A. 94, 1396-1401 (1997).</li><li>Shankar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+function+of+circulating+HIV-specific+CD8(+)+T+cells+in+chronic+human+immunodeficiency+virus+infection.">Impaired function of circulating HIV-specific CD8(+) T cells in chronic human immunodeficiency virus infection.</a> Blood. 96, 3094-3101 (2000).</li><li>Ward, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+Vpr+triggers+natural+killer+cell-mediated+lysis+of+infected+cells+through+activation+of+the+ATR-mediated+DNA+damage+response.">HIV-1 Vpr triggers natural killer cell-mediated lysis of infected cells through activation of the ATR-mediated DNA damage response.</a> PLoS Pathog. 5, e1000613-e1000613 (2009).</li><li>Ward, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+modulates+the+expression+of+ligands+important+in+triggering+natural+killer+cell+cytotoxic+responses+on+infected+primary+T-cell+blasts.">HIV modulates the expression of ligands important in triggering natural killer cell cytotoxic responses on infected primary T-cell blasts.</a> Blood. 110, 1207-1214 (2007).</li><li>Bonaparte, M. I., Barker, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killing+of+human+immunodeficiency+virus-infected+primary+T-cell+blasts+by+autologous+natural+killer+cells+is+dependent+on+the+ability+of+the+virus+to+alter+the+expression+of+major+histocompatibility+complex+class+I+molecules.">Killing of human immunodeficiency virus-infected primary T-cell blasts by autologous natural killer cells is dependent on the ability of the virus to alter the expression of major histocompatibility complex class I molecules.</a> Blood. 104, 2087-2094 (2004).</li><li>Stinchcombe, J. C., Griffiths, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretory+mechanisms+in+cell-mediated+cytotoxicity.">Secretory mechanisms in cell-mediated cytotoxicity.</a> Annu Rev Cell Dev Biol. 23, 495-517 (2007).</li><li>O'Doherty, U., Swiggard, W. J., Malim, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunodeficiency+virus+type+1+spinoculation+enhances+infection+through+virus+binding.">Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding.</a> J Virol. 74, 10074-10080 (2000).</li><li>Alter, G., Malenfant, J. M., Altfeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD107a+as+a+functional+marker+for+the+identification+of+natural+killer+cell+activity.">CD107a as a functional marker for the identification of natural killer cell activity.</a> J Immunol Methods. 294, 15-22 (2004).</li><li>Fogli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysis+of+endogenously+infected+CD4++T+cell+blasts+by+rIL-2+activated+autologous+natural+killer+cells+from+HIV-infected+viremic+individuals.">Lysis of endogenously infected CD4+ T cell blasts by rIL-2 activated autologous natural killer cells from HIV-infected viremic individuals.</a> PLoS Pathog. 4, e1000101-e1000101 (2008).</li><li>Tomescu, C., Chehimi, J., Maino, V. C., Montaner, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NK+cell+lysis+of+HIV-1-infected+autologous+CD4+primary+T+cells:+requirement+for+IFN-mediated+NK+activation+by+plasmacytoid+dendritic+cells.">NK cell lysis of HIV-1-infected autologous CD4 primary T cells: requirement for IFN-mediated NK activation by plasmacytoid dendritic cells.</a> J Immunol. 179, 2097-2104 (2007).</li><li>Ward, J. P., Bonaparte, M. I., Barker, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA-C+and+HLA-E+reduce+antibody-dependent+natural+killer+cell-mediated+cytotoxicity+of+HIV-infected+primary+T+cell+blasts.">HLA-C and HLA-E reduce antibody-dependent natural killer cell-mediated cytotoxicity of HIV-infected primary T cell blasts.</a> Aids. 18, 1769-1779 (2004).</li></ol>

Doğru yapıldığı zaman bu protokolü açıklanan Testler karşı degranüle ve HIV-1 enfekte olmuş hücreleri (bkz. Şekil 5) lyse NK hücreleri yeteneğini temsil eden bir resim sağlamalıdır. HIV ile enfekte olan hücreleri ve NK hücreleri lizis HIV ile enfekte olan hücreleri yanıt NK hücre degranülasyonu 10 doğru orantılı olmalıdır. HIV ile enfekte hücreleri sitotoksik tepkilerini ölçmek için iki NK hücre fonksiyonel testleri için güvenilir sonuçlar son derece NK hücrelerinin yanı sıra, son derece saf bir nüfusun enfekte hücreleri saflaştırılmış izolasyonu bağlıdır. Saflaştırılmış sahip NK hücreleri ve HIV-1 ile enfekte hücreler oldukça doğru efektör hücre oranı hedefi, başarı için kritik öneme sahiptir. Benzer şekilde, ölü ve apoptotik hücreleri hedef hücre popülasyonlarının kaldırılması efektör hücreleri ile 51 Cr etiketleme veya inkübasyon öncesinde önemli 51 Cr izotop etiketleme adım sırasında ölü ya da apoptotik hücrelerin varlığı olarak apoptoz gören hücreler tarafından içselleştirilmiş olabilir yüksek spontan sürümde neden olacak ve hesaplanan% özel lizis bozar. Ayrıca, ölü ya da apoptotik hücrelerin varlığı, CD107a ifade anormal derecede yüksek düzeyleri NK hücre degranülasyonu tetikleyebilir. Hassas pipetleme 51 Cr serbest tayinlerde, NK hücreleri ve hedef hücrelerin kuluçka aşağıdaki hücre süpernatantlar çıkarırken, gerekli her çoğaltmak kaldırıldı süpernatant hacmi farklılıkları gibi yüksek standart sapma neden olacaktır . Değişiklikler önce belirli reseptörleri veya ligandlar 6,11 antagonist antikorlar ile ko-kültür effektör veya hedefleri kuluçka NK hücreleri, HIV ile enfekte olan hücreleri lizis tetiklenmesi belirli NK reseptörleri rolünü değerlendirmek için bu protokoller yapılabilir . Sitokin tedavi edilen NK hücreleri (örneğin, IL-2, IL-15), NK hücreleri uyarılır 12 işlevselliğini değerlendirmek için kullanılabilir. Benzer şekilde, bu protokolleri kullanarak antikor bağımlı hücre sitotoksisite testleri yapılabilir. Anti-HIV antikorların (örneğin, anti-gp120), NK hücre düşük afinite Fc reseptörü CD16 13 tarafından tanınması için hedef hücrelere eklenebilir . Bu deneyleri, HIV ile enfekte olan hücreleri NK hücre sitotoksik tepkileri ölçmek için kurulmuş olmasına rağmen aynı zamanda NK hücreleri, HIV ile enfekte hücre tanıma aşağıdaki sitokin üretme yeteneğini ölçmek için modifiye edilebilir. Biz bu protokolü in vitro enfekte olan hücreleri hedef hücreler olarak kullanımı tanımlanmış olsa da biz son nesil açıklanan hedef hücrelerin HIV ile enfekte olan hastaların var . Bu izolasyon CD4 + T-hücrelerinin hücrelerin genişlemesi, interlökin-2 (11) varlığında mitojenler ile uyarılmasını takiben bir iki hafta boyunca takip gerektirir. Genişleme iki haftalık bir süre sonra, bu makalede anlatılan protokoller hedef hücreleri izole etmek için kullanılır.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Vaccutainer Tubes (Sodium Heparin)</td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>367874</td>
<td>&nbsp;</td>
<tr>
<td>RosetteSep CD4+ T-cell Isolation Kit</td>
<td>&nbsp;</td>
<td>StemCell Technologies</td>
<td>15062</td>
<td>&nbsp;</td>
<tr>
<td>CMF PBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30256.01</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>10437-028</td>
<td>&nbsp;</td>
<tr>
<td>Lymphocyte Separation Medium</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>25-072-CV</td>
<td>&nbsp;</td>
<tr>
<td>RPMI-1640</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30096.01 </td>
<td>&nbsp;</td>
<tr>
<td>Penicillin / Streptomycin</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SV30010</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>25-005-Cl</td>
<td>&nbsp;</td>
<tr>
<td>T-cell Expansion Kit</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>130-091-441</td>
<td>&nbsp;</td>
<tr>
<td>CMF HBSS (1x)</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30588.01</td>
<td>&nbsp;</td>
<tr>
<td>0.5M EDTA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>46-034-Cl</td>
<td>&nbsp;</td>
<tr>
<td>NK Cell Negative Isolation Kit</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>130-092-657</td>
<td>&nbsp;</td>
<tr>
<td>IMDM</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>12440-046</td>
<td>&nbsp;</td>
<tr>
<td>CD4+ Positive Isolation Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>113.31D</td>
<td>&nbsp;</td>
<tr>
<td>Dead Cell Removal Kit</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>130-090-101</td>
<td>&nbsp;</td>
<tr>
<td>Polybrene</td>
<td>&nbsp;</td>
<td>Santa Cruz</td>
<td>sc-134220</td>
<td>&nbsp;</td>
<tr>
<td>CD3 PacificBlue</td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>558117</td>
<td>&nbsp;</td>
<tr>
<td>CD14 PacificBlue</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>325616</td>
<td>&nbsp;</td>
<tr>
<td>CD20 PacificBlue</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>302328</td>
<td>&nbsp;</td>
<tr>
<td>CD56 APC</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>318310</td>
<td>&nbsp;</td>
<tr>
<td>CD69 PE</td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>555531</td>
<td>&nbsp;</td>
<tr>
<td>CD107a FITC</td>
<td>&nbsp;</td>
<td>Becton Dickenson</td>
<td>555800</td>
<td>&nbsp;</td>
<tr>
<td>51Chromium</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEZ030002MC</td>
<td>&nbsp;</td>
<tr>
<td>Gamma Counter Tubes</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>1270-401</td>
<td>&nbsp;</td>
<tr>
<td>2470 Automatic Gamma Counter</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>2470-0050</td>
<td>&nbsp;</td>
<tr>
<td>FACS Diva</td>
<td>&nbsp;</td>
<td>Beckton Dickenson</td>
<td>643629</td>
<td>&nbsp;</td>
<tr>
<td>FlowJo</td>
<td>&nbsp;</td>
<td>TreeStar</td>
<td>FJ-9-1YR</td>
<td>&nbsp;</td>		</tbody>
		</table>
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preparation and use of hiv-1 infected primary cd4+ t-cells as target cells in natural killer cell cytotoxic assays

Doğal öldürücü (NK) hücreleri, virüsle enfekte olan hücreleri doğuştan gelen bağışıklık yanıtının hayati bir bileşendir. NK hücreleri, HIV ile infekte hücrelere karşı NK hücre fonksiyonu herhangi bir anormallik tespit, çünkü potansiyel olarak kendi sitolitik aktivitesini artıracak tedavilere yol açabilir HIV-1 enfekte hücreleri tanıma ve lyse yeteneğini anlamak için önemlidir. HIV ile enfekte olan birincil T-hücre patlamalar ziyade hedef hücreleri sitotoksisite deneyleri enfekte T-hücre hatları kullanmak için bir ihtiyaç vardır. T-hücre, hatta enfeksiyon olmadan, NK hücre lizis oldukça duyarlıdırlar. Ayrıca, hedef ve effektör hücre lizis neden olacaktır arasında uyumsuzlukları büyük doku uygunluk kompleksi sınıf önlemek için otolog birincil hücreler kullanmak için gerekli. Primer HIV ile enfekte olan hücreleri NK hücre sitolitik yanıtları değerlendiren ilk çalışmalar, enfekte olmuş hücreleri 1,2 önemli öldürme göstermek için başarısız oldu . Ancak, kullanarak, HIV-1 enfekte birincil NK hücre fonksiyonel analizde hedef hücreler olarak T hücreleri enfekte olmamış hücrelerdeki 3 kontamine varlığı nedeniyle zor olmuştur. Bu tutarsız enfekte hücrenin bulaşmamış hücre oranı donör NK hücre fonksiyonunu değişkenliği nedeniyle olmayabilir örnekleri arasında NK hücre öldürme varyasyon neden olacaktır. Bu nedenle, deneyler arasındaki hücre oranları 3,4 hedef efektör standardize etmek amacıyla bir saflaştırılmış enfekte hücrenin nüfusu ile çalışmak faydalı olacaktır. İşte biz, ölü ya da ölmekte olan hücrelerin 3-6 kaldırmak için, HIV-1 yeteneğini enfekte olmuş hücreleri ve ticari kitleri durumu aşağı modüle CD4 yararlanarak HIV-1 enfekte olmuş hücreleri, son derece saf bir nüfusun izolasyon göstermektedir. Saflaştırılmış enfekte birincil T-hücre patlamalar sonra nüfus olarak 5-7 efektör NK hücreleri saflaştırılmış bir degranülasyonu veya sitotoksik tahlil ya da hedef olarak kullanılabilir. NK hücreleri degranülasyonu tayininde effektör olarak kullanın 8 &quot;sitolitik granül&quot; olarak adlandırılan uzman lizozomlar litik içeriğini serbest bırakmak için bir NK hücre yeteneğini değerlendirir. Florokrom CD107a sitolitik granül plazma membranına sigorta NK hücre yüzeyinde eksprese olan bir lizozomal membran proteini karşı konjuge antikor ile boyanarak hücre tanıma hedef yanıt degranüle NK hücrelerinin yüzde belirleyebilirsiniz. Alternatif olarak, NK hücre litik aktivite, hedef hücreleri, NK hücreleri varlığı hedef hücre içinde 51 Cr salınımı ile parçalanmış yüzdesi tayini için izin veren bir sitotoksik tayininde değerlendirilebilir.

Watch this Scientific Journal Video about Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays at JoVE.com

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

Sitotoksisite deneyleri, HIV bulaşmış hücrelerin doğal öldürücü hücre litik tepkileri ölçmek için hedef hücreler saflığı ile sınırlıdır. Biz burada, HIV-1 yeteneğini CD4 aşağı modüle yararlanarak HIV-1 ile enfekte birincil T-hücre patlamaların son derece saf bir nüfusun izolasyon göstermektedir.

Hazırlanması ve kullanımı, HIV-1 Enfekte İlköğretim CD4 + T-Hücreleri Hedef Hücreler olarak Natural Killer Hücre Sitotoksik Tahliller

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

preparation-use-hiv-1-infected-primary-cd4-t-cells-as-target-cells

Research

JoVE Journal

Immunology and Infection

25.8K Görüntüleme.  Rush University Medical Center. Sitotoksisite deneyleri, HIV bulaşmış hücrelerin doğal öldürücü hücre litik tepkileri ölçmek için hedef hücreler saflığı ile sınırlıdır. Biz burada, HIV-1 yeteneğini CD4 aşağı modüle yararlanarak HIV-1 ile enfekte birincil T-hücre patlamaların son derece saf bir nüfusun izolasyon göstermektedir.

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

Introducing Shear Stress in the Study of Bacterial Adhesion

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

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

Bakteriyel Yapışma Çalışma Kesme Stres Tanıtımı

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

İmmünoloji ve Enfeksiyon

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

Primer insan T hücrelerinin piggyBac Transposon Sistem Modifikasyon

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Yapay Antijen Hücre (AAPC) Aracılı Aktivasyon ve Doğal Öldürücü T hücreleri genişletilmesi Presenting

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

Yeni Araçlar HIV-1 ile enfekte bireyler gelen Düzenleyici T hücreleri büyütmek için

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

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 Enfekte CD4 Doğuştan Algılama Değerlendirilmesi<sup&gt; +</sup&gt; Bir kullanılması plazmositoid dendritik hücreler tarafından T Hücreleri<em&gt; Ex vivo</em&gt; Co-kültür sistemi.

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

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Etkin Killer yapay antijen sunan hücreler (KaAPC)<em&gt; İn Vitro</em&gt; İnsan Antijen-spesifik T hücrelerinin tükenmesi

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

Fare Dalak Değişmeyen Doğal Öldürücü T Hücreleri İzolasyon ve Yaygınlaştırılması için optimize Yöntemi

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

HIV-1 ile infekte Canlı İlköğretim İnsan Makrofagların Ölçülen fagosom Göç ve Hız

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

İnsan Tüm Kan Natural Killer Hücre Parçalayıcı Faaliyet Akışı Sitometrik Analizi

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.