עבודה זו נתמכה על ידי דן דאנקן שיתופי מענק מחקר (CMB וEJS), לב, ריאה הלאומית, ודם מכון (US4HL081007), לוקמיה ולימפומה פרס אגודת מחקר הקליני Scholar (CMB), והמכון הלאומי לסרטן (RO1 CA06150816; EJS).

1. בידוד התא mononuclear (יום 0) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנס ספייק של מתאם luer נקבה לתוך פתח יציאה מחלק 20% מיחידת דם הטבורים, לצרף מזרק ולהסיר דם. העברת דם מופשר עד 20 מ&quot;ל של RPMI חמם בצינור צנטריפוגה מ&quot;ל 50. יש לשטוף את שקית דם טבורים עם 5 מ&quot;ל של RPMI ולהעביר לאותו הצינור צנטריפוגה. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה תאים במשך 10 דקות ב 400 x גרם. לשאוב supernatant. </li><li style=";text-align:right;direction:rtl"> Resuspend תאים ב20 מ&quot;ל של RPMI החם. תאי שכבה על גבי 15 מ&quot;ל של Lymphoprep בצינור צנטריפוגה 50 מ&quot;ל. צנטריפוגה 40 דקות @ 400 x גרמו. </li><li style=";text-align:right;direction:rtl"> לקצור את הממשק המכיל את תאי mononuclear ולשטוף ב20 מ&quot;ל של RPMI. צנטריפוגה במשך 10 דקות @ 450 x גרמו. </li><li style=";text-align:right;direction:rtl"> לשאוב supernatant ולשטוף ב20 מ&quot;ל של RPMI. ספירת תאים ולסובב במשך 5 דקות @ 400 x גרמו. </li><li style=";text-align:right;direction:rtl"> לשאוב תאי supernatant ו resuspend ב 5 X 10 6 תאים / מ&quot;ל של בירת Cellgenix תקשורת (סרום חינם) mononuclear. הסר 1 מ&quot;ל לדור EBV-LCL והכניס 15 המ&quot;ל centrifuge צינור. </li></ol> 2. דור תאים דנדריטים (החל ביום 0) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 2 מיליליטר הצלחת של תאים (כבר בשעת 5 x 10 6 תאים / מ&quot;ל של DC תקשורת) לגם לתוך צלחת 6-כן. השאר באינקובטור במשך 1-2 שעות. </li><li style=";text-align:right;direction:rtl"> לאחר 1-2 שעות, לשטוף את התאים שאינם חסידים על ידי שטיפת 3-4 פעמים עם PBS. לאסוף את התאים שאינם חסידים והקפאה. הוסף 2 מ&quot;ל / היטב של תקשורת DC המכילה 1000 U / מ&quot;ל ​​של IL-4 ו 800 U / mL GM-CSF. </li><li style=";text-align:right;direction:rtl"> 3-4 ימים לאחר DC ייזום, לחדש IL-4 ו-GM-CSF על ידי הוספת 100 μL / גם של תקשורת המכילה ריכוז סופי של 1000U/mL IL-4 ו 800 U / mL GM-CSF. </li><li style=";text-align:right;direction:rtl"> ביום 5-6, הקציר DC על ידי גירוד היטב עם פיפטה העברה. ספירת התאים וצנטריפוגות הגדולים במשך 5 דקות @ 400 x גרם. Aspirate התקשורת וResuspend DC ב2x10 6 תאים / מ&quot;ל של DC תקשורת. הוסף 0.5 מ&quot;ל / היטב של צלחת 24 היטב. </li><li style=";text-align:right;direction:rtl"> Transduce DC באמצעות Ad5f35pp65 הווקטור במשרד פנים לכל תא של 10 יחידות זיהומיות. הוסף vector לכל אחד גם בצלחת 24 היטב. דגירת תאים עבור 1.5 שעות. לאחר 1.5 שעות, להוסיף 1.5 מ&quot;ל / היטב של DC מדיה מכיל: 1000 U / mL IL-4, 800 U / mL GM-CSF, 10 ng / mL TNF-אלפא, מיקרוגרם 1 / המ&quot;ל PGE-1, 100 ng / mL IL-6, ו 10 ng / mL IL-1beta. ציטוקינים אלו יבשילו את DCS. </li></ol> 3. דור של EBV-LCL (החל ביום 0) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צנטריפוגה שפופרת המכילה את 5 x 10 6 תאי mononuclear. הוסף 200 μL של EBV המרוכז B95.8 supernatant ו -1.8 מ&quot;ל של מדיה מלאה (RPMI + 10% FBS + 2 מ&quot;מ L-גלוטמין) ציקלוספורין המכיל (מיקרוגרם 1 / מ&quot;ל). </li><li style=";text-align:right;direction:rtl"> 100 μL aliquot של תאים לתוך 10 בארות של צלחת 96 היטב ו200 μL לתוך 5 בארות. הוסף 100 μL של מדיה המלאה + ציקלוספורין לבארות המכילות רק 100 μL של תאים. מלא בארות שנותרו עם מים סטריליים. </li><li style=";text-align:right;direction:rtl"> להאכיל LCL השבועי ולהרחיב מצלחת 96-היטב לצלחת 24 גם לאחר ~ 2 שבועות. אחרי שבוע נוסף, להעביר לבקבוק וT25 בהעברת השבוע לאחרT75 בקבוק. בשלב זה לא מומלץ להקפיא aliquots של LCL. זה בדרך כלל לוקח חודש 1 כדי לייצר כמות מספקת של LCL. </li></ol> 4. CTL ייזום - 7 ימים לאחר תחילת DCS <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> DCS הקציר ולהקרין בקצב של 30 Gy. שטוף 4 X וספירה. Resuspend DC בתקשורת סלולרית T המכיל סרום אדם (45% RPMI, לחץ עליה של 45%, 10% נסיוב אדם, 2 מ&quot;מ L-גלוטמין) @ 1 x 10 5 DCS / מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> הפשר תאים שאינם חסידים מצעד 2.2. שטוף תאים, ולספור. Resuspend ב 2 x 10 6 תאים שאינם חסידים / מ&quot;ל בתקשורת סלולרית T המכיל סרום אנושי. הוסף 10 ng / mL IL-7 ו-IL-12, ו 5 ng / mL IL-15. הוסף 1 מ&quot;ל של תאים לטובים בצלחת 24 היטב. הוסף 1 מ&quot;ל של בירה לטובה. מלא בארות ריקות של צלחת 24 היטב עם מים סטריליים. </li><li style=";text-align:right;direction:rtl"> 7 ימים לאחר תחילת תא T, הזנה ו / או לפצל את התאים עם תקשורת סלולרית T המכיל סרום אנושי. </li><li style=";text-align:right;direction:rtl"> 9-12 ימים לאחר תחילת תא T, להקפיא את תאי T עד LCL מוכן. </li><li style=";text-align:right;direction:rtl"> ONCE LCL הרחיב וpassaged בT75 צלוחיות, transduce LCL ידי צנטריפוגה במשך 5 דקות @ 400 x גרמו. הוסף את הווקטור לתא הגלול במשרד פנים של 100 יחידות זיהומיות לכל תא. דגירה במשך 1.5 שעות. לאחר 1.5 שעות, resuspend בתקשורת מלאה ב5 X 10 5 LCL / מ&quot;ל ולהוסיף 2 מ&quot;ל לכל גם בצלחת 24 גם 12. </li><li style=";text-align:right;direction:rtl"> לאחר 2 ימים, לקצור LCL ולהקרין ב40 Gy. שטוף 4 X וספירה. Resuspend LCL בתקשורת סלולרית T המכיל סרום אדם ב 2.5 10 5 תאים / מ&quot;ל x. הוסף 1 מ&quot;ל של LCL להיטב של צלחת 24 היטב. כמו כן, הוסף 5 X 10 6 LCL למכשיר Grex40 תרבות, כמו גם 5 ng / mL IL-15 13. </li><li style=";text-align:right;direction:rtl"> לקצור את תאי T. צנטריפוגה במשך 5 דקות @ 400 XG, לשאוב supernatant, resuspend ובתקשורת סלולרית T המכיל סרום אנושי ב1 x 10 6 תאים / מ&quot;ל T. הוסף 5 ng / mL IL-15 ולהוסיף 1 מ&quot;ל / היטב של תאי T (סך של 1 10 x 6 תאי T) לLCL שכבר נמצאים בצלחת 24 היטב. בGrex40, יביאהנפח הכולל של תקשורת עד 30 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> בפוסט גירוי 3-4 ימים, אז אם התאים הם confluent לפצל אותם (בצלחת) 1:1 ולהוסיף תקשורת טריה המכילה 50 U / mL IL-2. אם הם לא מתלכדים אז פשוט לשאוב ½ התקשורת ולהחליף עם מדיה המכילה 50 U / mL IL-2. בGrex40, לשאוב חצי מהתקשורת, להחליף עם תקשורת טריה, ולהוסיף 50 U / mL IL-2. </li><li style=";text-align:right;direction:rtl"> ביום 7, חוזר כמו בשלב 4.6 אלא להשתמש IL-2 ביום הגירוי, לא IL-15. </li></ol> 5. נציג תוצאות סכמטי של פרוטוקול ה-FDA ייצור GMP התואם מתואר באיור 1. התהליך לוקח כ 50 ימים. שיטות אופייניות של יצירת תאי T ספציפיים לוירוס להרחיב תאי T זיכרון קיים, עם זאת, חסרי דם טבורים ותאי T-וירוס מנוסה ולכן אנחנו צריכים vivo ממשלה הנאיבית T תאים לשעבר. לשם כך, אנו משתמשים בתאים דנדריטיים, כמו גם את הציטוקינים IL-7, IL-12, ו-IL-15,הכרחי ליצירה סגולית נגיפית. לאחר 3 גירויים, התשואה צריכה להיות מעל 100x10 6 תאים. אם תאים מספיקים אינם זמינים, גירויים נוספים יכולים להתבצע עד המספר הרצוי של CTLs זמין. רוב התאים אלה צריכים להיות CD3 + עם תערובת של CD4 + ותאי T CD8 +. לא צריך להיות פחות מ 15% תאי NK (CD3-/CD56 +) ופחות מ 1% CD19 + B תאים (איור 2). CTL המורחב צריך להכיר את האנטיגן pp65 מCMV, hexon ופנטון מאדנווירוס, כמו גם אנטיגנים EBV רבים שבאים לידי ביטוי בEBV-LCL. כאשר נבדק בבדיקת ELISPOT, CTL צריך להפריש יותר IFN-? בתגובה לאנטיגנים אלה מאשר אנטיגנים רלוונטיים (איור 3). CTL צריך גם lyse מטרות נגיפיות פפטיד פעם תקיעות PHA כאלה. בassay Cr 51 שחרור, CTL צריך lyse LCL, CMVpp65 פעם וadenovirus hexon ו /או מטרות פנטון פעם אבל לא פעם יעדים עם פפטידים רלוונטיים (איור 4). <img src="/files/ftp_upload/3627/3627fig1.jpg" alt="איור 1."/> איור 1. דור של CTL רב וירוס ספציפי מדם הטבורי. סכמטי המציג את כל התהליך של ייצור CTL מדם הטבורי. ראשית את תאי דם טבורי mononuclear מבודדים מחלק 20% מיחידת דם הטבורים המופרדות. למעט 5x10 6 תאים הנשמרים לדור LCL, כל התאים אז מצופים בתקשורת סלולרית הדנדריטים במשך 1-2 שעות, ובשלב הזה התאים שאינם חסידים נקצרים וקפוא. DC אז האכיל DC מדיה המכילה IL-4 ו-GM-CSF. לאחר 5 ימים של התרבות, DC הם התבגרו וtransduced עם וקטור adenoviral המכיל את האנטיגן pp65 immunodominant CMV. בייזום, DC אלה בשילוב עם התאים שאינם חסידים כמו גם ציטוקינים IL-7, IL-12,ו IL-15. בגירויים שלאחר מכן, באותו הווקטור adenoviral משמש לtransduce EBV-LCL, המשמשים כתאי מציגי אנטיגן. IL-15 משמשים בגירוי השני וIL-2 לאחר מכן. <img src="/files/ftp_upload/3627/3627fig2.jpg" alt="איור 2."/> איור 2. הפנוטיפ של כתוצאה מתאי T. הראה הוא אחוז תאי חיים הנכללים בשער הלימפוציטים. CTL הוא CD3 + וסוג CD4 + או CD8 + אבל בעיקר CD3-/CD56- וCD19-. <img src="/files/ftp_upload/3627/3627fig3.jpg" alt="איור 3."/> איור 3. פונקציונלי תא T. תא T ספציפי נבדק על ידי IFN-γELISPOT. תאי T שפעמו עם פפטידים חופפים פורשים כל החלבון של hexon, פנטון, pp65, ואנטיגן הרלוונטי IE-1. CTL לבד מציין תקשורת בלבד. Autologous LCL היה מוקרן והוסיף ב1x10 5 / כן. שמוצג הוא המקום המרושע להרכיב התא גount בארות שלושה עותקים. <img src="/files/ftp_upload/3627/3627fig4.jpg" alt="איור 4."/> איור 4. פעילות Cytolytic של CTL. היכולת של CTL כתוצאה lyse למטרות מבטאות אנטיגנים נגיפיים נבדקה בבדיקת 51 Cr. תקיעות 51 Cr הכותרת-PHA עצמיות היו פעמה עם פפטידים חופפים פורשים כל אנטיגן או 51 Cr-השתלת LCL הכותרת היה cocultured עם CTL. לאחר 4 שעות, שחרור גמא נספר על דלפק גמא.

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Hematol. 91, 588 (2010).</li><li>Einsele, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infusion+of+cytomegalovirus+(CMV)-specific+T+cells+for+the+treatment+of+CMV+infection+not+responding+to+antiviral+chemotherapy.">Infusion of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy.</a> Blood. 99, 3916 (2002).</li><li>Bao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+cytomegalovirus+pp65+and+IE-1+specific+cytotoxic+T+lymphocytes+for+cytomegalovirus-specific+immunotherapy+following+allogeneic+stem+cell+transplantation.">Expansion of cytomegalovirus pp65 and IE-1 specific cytotoxic T lymphocytes for cytomegalovirus-specific immunotherapy following allogeneic stem cell transplantation.</a> Biol. Blood Marrow Transplant. 14, 1156 (2008).</li><li>Shpall, E. J., Bollard, C. M., Brunstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+cord+blood+transplant+therapies.">Novel cord blood transplant therapies.</a> Biol. Blood Marrow Transplant. 17, S39-S45 (2011).</li><li>Vera, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+production+of+antigen-specific+T+cells+for+preclinical+and+clinical+applications+using+gas-permeable+rapid+expansion+cultureware+(G-Rex.">Accelerated production of antigen-specific T cells for preclinical and clinical applications using gas-permeable rapid expansion cultureware (G-Rex.</a> J. Immunother. 33, 305 (2010).</li></ol>

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<html> אסטרטגיות נוכחיות נועדו לשלוט זיהומים נגיפיים לאחר CBT יכולות להיות יעילות, אבל הם קשורים עם רעילויות משמעותיות, הם יקרים, ואינם מעניקות הגנה לטווח ארוכה מפני זיהום מאוחר יותר. למעשה, השימוש בכמה תרופות אנטי עלול להגביל את ההתפשטות של תאים ספציפיים לוירוס T שאחרת ה?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגים </td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 21870-076 </td></tr><tr><td> DC תקשורת </td><td> CellGenix </td><td> 20801-0500 </td></tr><tr><td> EHAA (הבינוני של לחץ) </td><td> אירווין מדעי </td><td> 9195 </td></tr><tr><td> סרום אדם </td><td> ג&#39;מיני מוצרים ביו </td><td> 100-110 </td></tr><tr><td> חדירות Cultureware 18 </td><td> וילסון-וולף </td><td> 80040S </td></tr><tr><td> IL-2 </td><td> כירון (TCH רוקחות) </td><td></td></tr><tr><td> IL-12 </td><td> NCI / CTEP </td><td></td></tr><tr><td> IL-15 </td><td> CellGenix </td><td> 1013-050 </td></tr><tr><td> IL-7 </td><td> מו&quot;פ </td><td> AFL207 </td></tr><tr><td> IL-1beta </td><td> מו&quot;פ </td><td> AFL201 </td></tr><tr><td> IL-6 </td><td> תאGenix </td><td> 1004-050 </td></tr><tr><td> GM-CSF </td><td> TCH מרקחת </td><td></td></tr><tr><td> IL-4 </td><td> מו&quot;פ </td><td> <a href="http://www.rndsystems.com/pdf/AFL204.pdf" target="_blank">AFL204</a> </td></tr><tr><td> TNF-אלפא </td><td> מו&quot;פ </td><td> AFL210 </td></tr><tr><td> Ad5f35pp65 </td><td> מתקן ייצור וקטור CAGT BCM </td><td></td></tr><tr><td> העברת פלזמה מוגדרת עם מתאם luer נשי </td><td> אמנה רפואית </td><td> 89-550-66j </td></tr><tr><td> Lymphoprep </td><td> Nycomed </td><td> 1114550 </td></tr></tbody></table>

expanding cytotoxic t lymphocytes from umbilical cord blood that target cytomegalovirus, epstein-barr virus, and adenovirus

הידבקות בוירוסים לאחר השתלת תאי גזע הן בין הגורמים השכיחים ביותר למוות, במיוחד לאחר דם טבורים (CB) השתלה (CBT) בי CB אינו מכיל מספרים ניכרים של תאי וירוס מנוסים T שיכול להגן על הנמען מהזיהום 1. - 4 אנחנו ואחרים הראינו שנוצרו וירוס הספציפי CTL מתורמים נושאים את ה HIV והחדירו לנמען הם בטוחים ומגוננים. 5-8 עם זאת, עד לאחרונה, תאי T ספציפיים לוירוס לא יכולים להיות שנוצרו מדם הטבורי, כנראה עקב היעדר תאי T זיכרון ספציפיים לוירוס. במאמץ טוב יותר כדי לחקות את התנאים בייזום vivo של תאי T נאיביים, הקים שיטה שהשתמשה בתאים דנדריטיים CB-derived (DC) transduced עם וקטור adenoviral (Ad5f35pp65) המכיל את האנטיגן pp65 immunodominant CMV, ולכן נוהג סגולי תא T לקראת CMV ואדנווירוס. 9 בייזום, אנו משתמשים ma אלהDCS tured כמו גם תאי T CB-derived בנוכחותם של ציטוקינים IL-7, IL-12, ו IL-15. 10 בגירוי השני שהשתמשנו EBV הפך-תאי B, או EBV-LCL, שמביעים שניהם אנטיגנים EBV ממוסס סמוי ו. Ad5f35pp65-transduced EBV-LCL משמש כדי לגרות את תאי T בנוכחות של IL-15 בגירוי השני. גירויים שלאחר מכן להשתמש Ad5f35pp65-transduced EBV-LCL וIL-2. מ50x10 6 תאי mononuclear CB אנו מסוגלים לייצר למעלה מ 150 10 6 תאי T x וירוס ספציפיים שlyse מטרות אנטיגן פעמו וציטוקינים שחרור בתגובה לגירוי antigenic. 11 תאים אלו יוצרו באופן GMP תואם רק באמצעות חלק 20% מיחידת דם טבורים מופרדת ותורגם לשימוש קליני.

Watch this Scientific Journal Video about Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus at JoVE.com

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

כאן אנו מתארים אימון הראשון הטוב הייצור (GMP)-תואמת שיטה של ​​ייצור תאי T ציטוטוקסיים וירוס ספציפיים (CTL) מדם טבורים, מקור לתאי T בעיקר נאיביים.

הרחבת לימפוציטים מסוג T ציטוטוקסיים מדם טבורים שציטומגלווירוס יעד, וירוס אפשטיין באר, וadenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

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Research

JoVE Journal

Immunology and Infection

12.7K Views.  Baylor College of Medicine. כאן אנו מתארים אימון הראשון הטוב הייצור (GMP)-תואמת שיטה של ​​ייצור תאי T ציטוטוקסיים וירוס ספציפיים (CTL) מדם טבורים, מקור לתאי T בעיקר נאיביים.

Video: Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

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

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 cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate 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 ...

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.

שיטות להעריך מתווכת בטא מוות של תאים על ידי לימפוציטים מסוג T ציטוטוקסיות

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

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.

היכרות עם מתח שאר בחקר הדבקה חיידקית

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

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

הקמת Epstein-Barr Virus צמיחה הפך שורות תאים Lymphoblastoid

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

יישום קליני של<em&gt; יפהפה נרדם</em&gt; ומלאכותי תאי מציגי אנטיגן גנטי לשינוי תאי T מדם טבורים היקפי וטבורים

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

בידוד של קבוצות משנה מבשר B-cell מדם טבורים

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

בידוד ובידול של Th17 הנאיביים T מסוג CD4 לימפוציטים

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Development of an IFN-&amp;#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

פיתוח של חסינות ELISPOT Assay להעריך γ-IFN אבעבועות רוח, זוסטר וירוס ספציפי לתא בתיווך לאחר השתלת דם טבורים

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

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.

דור של תאי T האנושי Alloantigen ספציפיים מהדם היקפי

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

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

מחוגי ניידות ותפקוד ציטוטוקסיות של retroviral מעתיקה וקטור transduced, שאינם חסיד Alloresponsive T לימפוציטים

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...