<ol>
	<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>

אין ניגודי אינטרסים הכריז.

כאשר נעשה בצורה נכונה מבחני המתואר בפרוטוקול זה אמור לספק תמונה מייצגת של יכולת התאים נ&quot;ק כדי degranulate כנגד lyse HIV-1 תאים נגועים (ראה איור 5). Degranulation תא NK בתגובה ל-HIV נגועים התאים לתאי NK-HIV נגועים בתאי תמוגה צריך להיות ביחס ישר 10. תוצאות אמינות עבור שני מבחני תא NK פונקציונלי למדוד את התגובות ציטוטוקסיות ל-HIV נגועים בתאי תלויים הבידוד של מטוהרים מאוד תאים NK, כמו גם אוכלוסיה מטוהרים מאוד של תאים נגועים. לאחר מטוהרים בתאי NK ו - HIV-1 תאים נגועים הם קריטיים להשגת מפעיל מדויק למדי ליעד יחס התא. כמו כן, הסרת תאים מתים אפופטוטיים מן אוכלוסיות תאים יעד חשוב לפני תיוג Cr 51 או דגירה עם התאים מפעיל. Cr 51 עשוי להיות מופנמת על ידי התאים עוברים אפופטוזיס כמו נוכחות של תאים מתים או אפופטוטיים במהלך השלב תיוג איזוטופ תגרום לשחרור ספונטני גבוהה יהיה לעוות את תמוגה% מחושב ספציפית. יתר על כן, נוכחותם של תאים מתים או אפופטוטיים עלולה לעורר degranulation תא NK וכתוצאה מכך רמות גבוהות באופן חריג של הביטוי CD107a. Pipetting מדויק הוא הכרחי בעת הסרת תאים ללא supernatants הבאים הדגירה של תאים NK ותאי היעד של 51 מבחני Cr לשחרר, כמו הבדלים בהיקף supernatant להסיר לשכפל כל טוב תגרום סטיות תקן גבוהות. שינויים ניתן לבצע הפרוטוקולים הללו כדי להעריך את התפקיד של קולטנים ספציפיים NK מפעילה תמוגה NK של HIV לתאים תאים הנגועים על ידי דוגרים effectors או מטרות לקראת תרבות בשיתוף עם נוגדנים אנטגוניסטית לרצפטורים ספציפיים או ligands 6,11. ציטוקין שטופלו בתאי NK (למשל, IL-2, IL-15) ניתן להשתמש כדי להעריך את התפקוד של תאים NK מגורה 12. כמו כן, תלוי נוגדן תא מבחני cytotoxicity ניתן לבצע בעזרת הפרוטוקולים הללו. נוגדנים נגד HIV (למשל, אנטי gp120) ניתן להוסיף את התאים היעד להכרה על ידי קולטן תא NK נמוך Fc זיקה CD16 13. מבחני אלה, למרות שהוקם כדי למדוד תגובות התא NK ציטוטוקסיות ל-HIV בתאים נגועים, יכול גם להיות שונה כדי למדוד את היכולת של התאים לייצר ציטוקינים NK הבאים הכרה HIV-נגוע התא. למרות שאנו המתואר פרוטוקול זה את השימוש בתאים במבחנה כמו תאים נגועים היעד שתיארנו לאחרונה את הדור של תאים היעד של חולים עם HIV. זה מצריך את בידודו של CD4 + T-תאים ואחריו התפשטות של התאים על פני תקופה של שבועיים בעקבות גירוי עם mitogens בנוכחות interleukin-2 11. לאחר תקופה שני בשבוע של התרחבות, הפרוטוקולים המתואר במאמר זה נעשה שימוש כדי לבודד את התאים היעד.

<table><tbody><tr><td>Vaccutainer Tubes (Sodium Heparin)</td><td>BD Biosciences</td><td>367874</td><td></td></tr><tr><td>RosetteSep CD4+ T-cell Isolation Kit</td><td>Stem Cell Technologies</td><td>15062</td><td></td></tr><tr><td>CMF PBS</td><td>Hyclone</td><td>SH30256.01</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>10437-028</td><td></td></tr><tr><td>Lymphocyte Separation Medium</td><td>Cellgro</td><td>25-072-CV</td><td></td></tr><tr><td>RPMI-1640</td><td>Hyclone</td><td>SH30096.01</td><td></td></tr><tr><td>Penicillin / Streptomycin</td><td>Hyclone</td><td>SV30010</td><td></td></tr><tr><td>L-glutamine</td><td>Cellgro</td><td>25-005-Cl</td><td></td></tr><tr><td>T-cell Expansion Kit</td><td>Miltenyi Biotec</td><td>130-091-441</td><td></td></tr><tr><td>CMF HBSS (1x)</td><td>Hyclone</td><td>SH30588.01</td><td></td></tr><tr><td>0.5M EDTA</td><td></td><td>46-034-Cl</td><td></td></tr><tr><td>NK Cell Negative Isolation Kit</td><td>Miltenyi Biotec</td><td>130-092-657</td><td></td></tr><tr><td>IMDM</td><td>GIBCO, by Life Technologies</td><td>12440-046</td><td></td></tr><tr><td>CD4+ Positive Isolation Kit</td><td>Invitrogen</td><td>113.31D</td><td></td></tr><tr><td>Dead Cell Removal Kit</td><td>Miltenyi Biotec</td><td>130-090-101</td><td></td></tr><tr><td>Polybrene</td><td>Santa Cruz Biotechnology, Inc.</td><td>sc-134220</td><td></td></tr><tr><td>CD3 PacificBlue</td><td>BD Biosciences</td><td>558117</td><td></td></tr><tr><td>CD14 PacificBlue</td><td>Biolegend</td><td>325616</td><td></td></tr><tr><td>CD20 PacificBlue</td><td>Biolegend</td><td>302328</td><td></td></tr><tr><td>CD56 APC</td><td>Biolegend</td><td>318310</td><td></td></tr><tr><td>CD69 PE</td><td>BD Biosciences</td><td>555531</td><td></td></tr><tr><td>CD107a FITC</td><td>BD Biosciences</td><td>555800</td><td></td></tr><tr><td>&lt;sup&gt;51&lt;/sup&gt;Chromium</td><td>PerkinElmer, Inc.</td><td>NEZ030002MC</td><td></td></tr><tr><td>Gamma Counter Tubes</td><td>PerkinElmer, Inc.</td><td>1270-401</td><td></td></tr><tr><td>2470 Automatic Gamma Counter</td><td>PerkinElmer, Inc.</td><td>2470-0050</td><td></td></tr><tr><td>FACS Diva</td><td>BD Biosciences</td><td>643629</td><td></td></tr><tr><td>FlowJo</td><td>Tree Star, Inc.</td><td>FJ-9-1YR</td><td></td></tr></tbody></table>

preparation and use of hiv-1 infected primary cd4+ t-cells as target cells in natural killer cell cytotoxic assays

טבעיים (NK) תאים הרוצח הם מרכיב חיוני של התגובה החיסונית המולדת תאים הנגועים בנגיף. חשוב להבין את היכולת של התאים נ&quot;ח להכיר lyse HIV-1 תאים נגועים כי זיהוי כל aberrancy בתפקוד התא NK נגד HIV תאים הנגועים העלול להוביל לטיפולים כי היה לשפר את פעילות cytolytic שלהם. יש צורך להשתמש ב-HIV נגועים העיקרי T-cell תקיעות כמו תאים היעד דווקא אז נגוע-T-cell שורות מבחני cytotoxicity. T-cell הקווים, גם ללא זיהום, רגישים מאוד תמוגה התא NK. יתר על כן, יש צורך להשתמש תאים ראשוניים עצמיים כדי למנוע histocompatibility העיקריים מורכבים בכיתה אני חוסר התאמה בין המטרה לבין תא מפעיל כי תגרום תמוגה. מחקרים מוקדמים הערכת התגובות תא NK cytolytic ל-HIV נגועים תאים ראשוניים לא הצליחו להראות הרג משמעותי של 1,2 התאים הנגועים. עם זאת, שימוש ב-HIV-1 נגוע העיקרי מתאי T כמו תאים היעד מבחני תא NK פונקציונלי כבר קשה בשל נוכחותם של מזהמים תאים נגוע 3. זה תא נגוע עקבי יחס תא נגוע תגרום וריאציה בהריגת התא NK בין דגימות כי לא יכול להיות בגלל השתנות בתפקוד התא NK התורם. לכן, זה יהיה מועיל לעבוד עם האוכלוסייה מטוהרים תא נגוע כדי לתקנן את מפעיל למקד יחס בין תא ניסויים 3,4. כאן אנו מדגימים את בידודה של אוכלוסייה מטוהרים של HIV-1 תאים נגועים על ידי ניצול היכולת של HIV-1 אל מטה לווסת CD4 על תאים נגועים ואת הזמינות של ערכות מסחריות כדי להסיר תאים מתים או גוססים 3-6. מטוהרים נגוע העיקרי T-cell תקיעות אז יכול לשמש מטרות degranulation או assay ציטוטוקסיות עם מטוהרים תאים NK כמו האוכלוסייה מפעיל 5-7. השימוש בתאי NK כמו effectors ב assay degranulation מעריך את יכולתו של התא NK לשחרר את התוכן ממס של lysosomes המקצועית 8 בשם &quot;גרגרי cytolytic&quot;. עד מכתים עם נוגדן מצומדות fluorochrome נגד CD107a, חלבון קרום lysosomal הופך הביע על פני התא NK כאשר גרגרי cytolytic הפתיל הממברנה פלזמה, אנו יכולים לקבוע איזה אחוז של תאים NK degranulate בתגובה היעד הכרה התא. לחילופין, פעילות NK תא ממס ניתן להעריך assay ציטוטוקסיות המאפשר קביעת אחוז התאים היעד lysed על ידי שחרור של 51 Cr מתוך תא המטרה בנוכחות תאים NK.

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

מבחני cytotoxicity למדוד טבעי התא הרוצח התגובות ממס ל-HIV נגועים בתאי מוגבל על ידי טוהר התאים היעד. אנו מדגימים כאן את בידודה של אוכלוסייה מטוהרים של HIV-1 נגוע T-cell תקיעות ראשונית על ידי ניצול היכולת של HIV-1 אל מטה לווסת CD4.

הכנה ושימוש של HIV-1 CD4 ראשי מאשרום + T-תאים כמו תאים היעד של טבע Cell מבחני Killer ציטוטוקסיות

 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

25.9K Views.  Rush University Medical Center. מבחני cytotoxicity למדוד טבעי התא הרוצח התגובות ממס ל-HIV נגועים בתאי מוגבל על ידי טוהר התאים היעד. אנו מדגימים כאן את בידודה של אוכלוסייה מטוהרים של HIV-1 נגוע T-cell תקיעות ראשונית על ידי ניצול היכולת של HIV-1 אל מטה לווסת CD4.

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

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

חזותי Cell ל-תא העברה של HIV באמצעות המשובטים פלורסנט של HIV מיקרוסקופיה confocal חיים

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

פיתוח נייד מסוג ספציפי נגד HIV gp120 aptamers למסירה siRNA

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

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.

Assay כמותית תפוקה גבוהה מתא בודד Cytotoxicity לתאי T

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

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.

במבחנה Assay כדי להעריך את ההשפעה של immunoregulatory מסלולים על פונקצית Effector הנייד HIV-ספציפית T מסוג CD4

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.

הערכת חישה המולדת של CD4 הנגוע ב- HIV-1<sup&gt; +</sup&gt; T תאים על ידי תאים דנדריטים Plasmacytoid שימוש<em&gt; Ex vivo</em&gt; עמית תרבות מערכת.

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

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

וזמינותו Cytotoxicity מבוססי Cytometry זרימה להערכה של פעילות תאי NK אנושי

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

שיטה פשוטה ויעילה להקים NK וקווים תא T של חולים עם דלקת כרונית פעילה וירוס אפשטיין בר

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

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.

תפירה Vivo ציטורעילות Assays ללמוד אימונומימינינס ב-CD8 התגובות הספציפיות לגידול של תאים

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

<meta charset="utf8"></head><body>חקר מתח ER והפעלת UPR במהלך זיהום HIV-1 בתאי T

Measuring Endoplasmic Reticulum Stress and Unfolded Protein Response in HIV-1 Infected T-Cells and Analyzing its Role in HIV-1 Replication

Viral infections can cause Endoplasmic Reticulum (ER) stress due to abnormal protein accumulation, leading to Unfolded Protein Response (UPR). Viruses have developed strategies to manipulate the host UPR, but there is a lack of detailed understanding of UPR modulation and its functional significance during HIV-1 infection in the literature. In this context, the current article describes the protocols used in our laboratory to measure ER stress levels and UPR during HIV-1 infection in T-cells and the effect of UPR on viral replication and infectivity. 
Thioflavin T (ThT) staining is a relatively new method used to detect ER stress in the cells by detecting protein aggregates. Here, we have illustrated the protocol for ThT staining in HIV-1 infected cells to detect and quantify ER stress. Moreover, ER stress was also detected indirectly by measuring the levels of UPR markers such as BiP, phosphorylated IRE1, PERK, and eIF2&#945;, splicing of XBP1, cleavage of ATF6, ATF4, CHOP, and GADD34 in HIV-1 infected cells, using conventional immunoblotting and quantitative reverse transcription polymerase chain reaction (RT-PCR). We have found that the ThT-fluorescence correlates with the indicators of UPR activation. This article also demonstrates the protocols to analyze the impact of ER stress and UPR modulation on HIV-1 replication by knockdown experiments as well as the use of pharmacological molecules. The effect of UPR on HIV-1 gene expression/replication and virus production was analyzed by Luciferase reporter assays and p24 antigen capture ELISA, respectively, whereas the effect on virion infectivity was analyzed by staining of infected reporter cells. Collectively, this set of methods provides a comprehensive understanding of the Unfolded Protein Response pathways during HIV-1 infection, revealing its intricate dynamics.

<meta charset="utf8"></head><body>מחבר זרקור: חקר התפקיד של תגובת חלבון נפרש בשכפול HIV-1 והדבקה

Here, we describe some established methods to determine endoplasmic reticulum (ER) stress and unfolded protein response (UPR) activation, with particular emphasis on HIV-1 infection. This article also describes a set of protocols to investigate the effect of ER stress/UPR on HIV-1 replication and virion infectivity.

מדידת לחץ רשתית אנדופלסמית ותגובת חלבון פתוחה בתאי T נגועים ב- HIV-1 וניתוח תפקידה בשכפול HIV-1

Viral infections can cause Endoplasmic Reticulum (ER) stress due to abnormal protein accumulation, leading to Unfolded Protein Response (UPR). Viruses ...