האס נתמך בחלקו על ידי HHMI המד לגראד ההדרכה גרנט באמצעות תכנית TBMM. MHW נתמך בחלקו על ידי פרס קריירת פיתוח מהמחלקה לענייני יוצאי הצבא ותמיכתה הנדיבה של ד&quot;ר והגב &#39;הרולד Selzman. עבודה זו נתמכה גם בחלקו על ידי המכון הלאומי לבריאות לימפומה נבג המענק P50CA126752 וNIH R01 DK093660.

יום 0 1. בידוד של PBMCs דם אנושי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> איסוף 20 מ&quot;ל של דם אדם טרי באמצעות venipuncture לתוך צינורות vacutainer Na-ההפרין. </li><li style=";text-align:right;direction:rtl"> דם תערובת והמתקדמת RPMI 1640 ב1:1 (v / v) יחס. </li><li style=";text-align:right;direction:rtl"> הוסף בינוני 20 מ&quot;ל lymphoprep לצינור צנטריפוגה 50 מ&quot;ל (25 מעלות צלזיוס). לאט 25-30 מיליליטר שכבה של 1640 תמהיל דם RPMI על גבי lymphoprep. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה XG ב 400 במשך 40 דקות ללא בלמים. </li><li style=";text-align:right;direction:rtl"> לאסוף שתי שכבות נפרדות ומטושטשות באמצעות פיפטה פנויה ל10 מ&quot;ל של 1x PBS (25 ° C) ולהביא את הנפח של עד 50 מ&quot;ל עם 1x PBS. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה ב XG 450 עבור 10 דקות. </li><li style=";text-align:right;direction:rtl"> לשאוב supernatant לחלוטין ולהוסיף 20 מ&quot;ל של המתקדם RPMI 1640. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה XG ב 400 למשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> לשאוב supernatant. הוסף 10 מ&quot;ל של תקשורת הסלולרית T המלאה בתוספת 5 ng / ml rhIL-15 prewarmed ב 37 ° C. </li><li style=";text-align:right;direction:rtl"> לספור את מספר התאים והצלחת בפרוות תרביות רקמה גם 24צלחת אד ב2 10 x 6 תאים / גם בתקשורת סלולרית T המלאה בתוספת 5 ng / ml rhIL-15 הוסף מים סטריליים לבארות סמוכות. דגירת הלילה בחממת humidified על 37 מעלות צלזיוס, 5% CO 2. </li></ol> יום 1 2. פלטות עם ציפוי אנטי-CD28 ואנטי CD3 נוגדן לגירוי תאי T <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דלל נוגדנים אנטי CD28 ואנטי CD3 במים סטריליים בריכוז של מיקרוגרם 1 / מ&quot;ל ​​כל אחד. </li><li style=";text-align:right;direction:rtl"> הוסף 500 μl פתרון כל נוגדנים ל 5 בארות מסומנות של תרבות רקמות שאינן מצופית 24 צלחת היטב. </li><li style=";text-align:right;direction:rtl"> הוסף מים סטריליים כדי לנוח מהבארות. עטוף את הצלחת בניילון ומקום פסיכולוג ב4 ° C מקרר. </li></ol> 3. Nucleofection של תאי T Unstimulated <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ° C ותוספת עם 5 ng / ml של rhIL-15 תקשורת הסלולרית T Prewarm ב 37. להכין תמיסת nucleofector מלאה על ידי הוספת 500 μl של מוסף Nucleofector 1-2.25 מ&quot;ל של פתרון Nucleofector. </li><li style=";text-align:right;direction:rtl">5 מיקרוגרם liquot כל אחד מtransposon (Zeo-PT-CMV-eGFP) וtransposase (pCMV-piggyBac) בצינור 1.5 מיליליטר microfuge הערה:. זה עשוי להיות נחוץ כדי לייעל transposase וכמות DNA transposon למסירת גן אופטימלית ולמזער רעילות סלולרית. את פלסמידים ניתן לקבל מהמחברים על ידי בקשה. </li><li style=";text-align:right;direction:rtl"> PBMCs אסיף מצלחת 24 גם תרבות הרקמה לתוך צינור מ&quot;ל 50. לספור את מספר התאים. שמור 2 10 x 6 תאים לשימוש כשליטה במהלך cytometry זרימה. </li><li style=";text-align:right;direction:rtl"> הוסף 07-10 אוקטובר 6 תאי x לצינור 15 מ&quot;ל וצנטריפוגה XG ב 400, 5 דקים &#39;, לשאוב supernatant ואצבע קפיצי הגלולה. </li><li style=";text-align:right;direction:rtl"> הוסף 100 μl של פתרון תא T מלא nucleofection לרופף תא גלול. </li><li style=";text-align:right;direction:rtl"> הוסף את תערובת פתרון תאים למבחנה המכילה את פלסמידים. </li><li style=";text-align:right;direction:rtl"> מוסיף את התערובת בתאי פלסמיד פתרון לחלק התחתון של קובט nucleofection. היזהר שלא להציג את כל בועות. </li><li style=";text-align:right;direction:rtl"> Nucleofect התאים באמצעות פרוגרם U-014 (תאי T, Unstimulated אדם, <a href="http://bio.lonza.com/resources/product-instructions/protocols/" target="_blank">http://bio.lonza.com/resources/product-instructions/protocols/).</a> </li><li style=";text-align:right;direction:rtl"> מייד להוסיף 500 μl של תקשורת prewarmed עם rhIL-15 לקובט. להעביר את התאים להיטב של צלחת גם 24 עם 1.5 מ&quot;ל של תקשורת עם prewarmed rhIL15. </li><li style=";text-align:right;direction:rtl"> דגירת הלילה בחממת humidified על 37 מעלות צלזיוס, 5% CO 2. </li></ol> יום 2 4. גירוי לא ספציפי של תאי T <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תאי קציר ולקבוע מספרים סלולריים. להפריש 0.5 x 10 6 תאים לזרימה cytometry כדי לקבוע את התדירות של תאי GFP חיוביים. השתמש בPBMCs הלא transfected כקבוצת ביקורת. </li><li style=";text-align:right;direction:rtl"> לשאוב את פתרון נוגדן CD3/28 מהצלחת שאינה רקמת התרבות המצופית ולשטוף היטב עם כל תקשורת סלולרית טי. </li><li style=";text-align:right;direction:rtl"> Resuspend תאי nucleofected ב0.5 10 x 6 תאים למ&quot;ל בתקשורת CTL המלאהבתוספת 5 ng / ml rhIL-15. הוסף 2.0 מ&quot;ל כל אחד מתאי nucleofected ב 4 בארות של צלחת תרבית הרקמה שאינן. הוסף 0.5 x 10 6 תאי nucleofected שאינם ל 5 כן. </li><li style=";text-align:right;direction:rtl"> דגירה במשך 3 ימים בחממת humidified על 37 מעלות צלזיוס, 5% CO 2. </li></ol> יום 5 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לקצור את תאי T מגורה מצלחת תרבית הרקמה שאינה המצופית. </li><li style=";text-align:right;direction:rtl"> לספור וreplate את התאים בתרבית רקמת צלחת גם מצופית 24 על 0.7 x 10 6 תאים / מ&quot;ל בתקשורת סלולרית T עם 5 ng / ml IL-15. </li></ol> יום 7 7. ניתוח של ביטוי גנים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תאי קציר וכתם עם CD8 נוגדן אנטי אנושי (אפשר גם להשתמש CD4 אנטי אנטי CD3 ו) ולנתח עם cytometry זרימה לביטוי של GFP%. </li></ol> יום 8 (אופציונלי) 8. התרחבות של תאי T <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ביום 8 לאחר transfection, תאי T ניתן replated בTמדיה סלולרית עם IL-15 בצפיפות של 0.7 x 10 6 תאים לכל גם להתרחבות נוספת 16. </li></ol>

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אין ניגודי האינטרסים הכריזו.

השיטה המתוארת כאן מאפשרת שינוי transgene יציב של לימפוציטים מסוג T האנושי ראשוני. בדקנו את השימוש במערכת transposon piggyBac עבר לשנות תאי T להביע גן כתב (יותר מ 4 שבועות), גן התאבדות אינו immunogenic, קולט אנטיגן chimeric לחיסון מאמצת (ליותר מ 100 ימים), ולהנדס התנגדות לתרופות מדכאות חיסון ...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם המגיב </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות (אופציונלי) </td></tr><tr><td> Lympholyte </td><td> Cedarlane </td><td> CL5015 </td><td></td></tr><tr><td> המתקדם RPMI 1640 </td><td> LifeTechnologies </td><td> 12633020 </td><td></td></tr><tr><td> סרום שור עוברי Hyclone </td><td> הפישר סיינטיפיק </td><td> SH3008803 </td><td></td></tr><tr><td> מוסף GlutaMAX- </td><td> LifeTechnologies </td><td> 35050-061 </td><td></td></tr><tr><td> IL-15 אנוש חלבון רקומביננטי </td><td> eBioscience </td><td> 14-8159 </td><td></td></tr><tr><td> קיט מקסי פלסמיד EndoFree </td><td> Qiagen </td><td> 12362 </td><td></td></tr><tr><td> Amaxa Nucleofector </td><td> Lonza </td><td> AAD-1001S </td><td> &amp; Nbsp; </td></tr><tr><td> תאי T אנושי Nucleofector קיט </td><td> Lonza </td><td> VPA-1002 </td><td></td></tr><tr><td> CD8-APC </td><td> דרום יוטק </td><td> 9536-11 </td><td></td></tr><tr><td> אנטי אנושי CD3 </td><td> eBioscience </td><td> 16-0037-81 </td><td></td></tr><tr><td> CD28 אנטי אנושי </td><td> BD Pharmingen </td><td> 555725 </td><td></td></tr><tr><td> פלייט מטפל בתרביות רקמה 24 ובכן </td><td> BD פלקון </td><td> 353047 </td><td></td></tr><tr><td> תרבית רקמה טובה ללא 24 מטופלי פלייט </td><td> BD פלקון </td><td> 351147 </td><td></td></tr><tr><td></td><td></td><td></td><td> תא T מלא מדיה רכב 1x המתקדם RPMI 1640 5% בסרום שור עוברי מומת חום 2 מ&quot;מ GlutamaxIM- </td></tr></tbody></table>

piggybac transposon system modification of primary human t cells

מערכת transposon piggyBac היא טבעי פעילה, המופק ממקור 1,2 כרוב Looper העש. מערכת שאינה נגיפית זה פלסמיד מבוסס, בדרך כלל ניצול שני פלסמידים עם אחד המבטא את אנזים transposase piggyBac וtransposon פלסמיד מטפח את הגן (ים) של עניין בין אלמנטים חוזרים הפוכים אשר נדרשים לפעילות העברת גנים. העברת גנים מתווך PiggyBac דרך מנגנון &quot;גזור ודבק&quot; לפי transposase משלב קטע transposon לתוך הגנום של תא המטרה (הים) של עניין. PiggyBac הוכיח פעילות מסירת גן יעילה במגוון רחב של חרקי 1,2, 3-5 יונקים, ו אדם cells6 לרבות תאי T אנושיים ראשוניים 7,8. לאחרונה, transposase piggyBac היפראקטיבי נוצר שיפור יעילות העברת גני 9,10. תאי T של אדם הם intere הקלינירח לחיסון מאמצת של 11 סרטן. מן ראוי לציין כי הניסוי הקליני הראשון הכולל שינוי transposon של תאי T אנושיים המשתמשים במערכת transposon היפיפייה הנרדמת אושר 12. יש לנו הערכה בעבר את השירות של piggyBac כהמתודולוגיה שאינה נגיפית הנדסה גנטית של תאי T אנושיים. מצאנו piggyBac להיות יעיל בשינוי הגנטי של תאי T אנושיים עם גן כתב וגן ההתאבדות מושרה אינו immunogenic 7. ניתוח של אתרי אינטגרציה הגנומי חשף חוסר ההעדפה להשתלבות או ליד אונקוגנים פרוטו ידוע 13. אנחנו השתמשנו piggyBac לימפוציטים גנים לשנות ציטוטוקסיות T לבצע קולט אנטיגן chimeric מופנה כנגד אנטיגן גידול HER2, וגילינו כי תאים ששונו גנטי T מתווכים סיכול ממוקדים של תאי גידול מסוג HER2 חיוביים במבחנת in vivo במודל עכבר orthotopic 14. השתמשנו גם piggyBacכדי לייצר תאי T אנושיים עמידים לrapamycin, אשר אמור להיות יעילים בטיפול בסרטן שבי rapamycin מנוצל 15. בזאת, אנו מתארים שיטה לשימוש piggyBac גנטי כדי לשנות את תאי T אנושיים ראשוניים. זה כולל בידוד של תאי דם היקפיים (mononuclear PBMCs) מדם של בני אדם ואחריו תרבות, שינוי גנטי, והפעלת תאי טי. לצורך הדו&quot;ח זה, תאי T שונו עם גן כתב (eGFP) לניתוח וכימות של ביטוי גנים על ידי cytometry זרימה. PiggyBac ניתן להשתמש כדי לשנות את תאי T אנושיים עם מגוון רחב של גנים של עניין. למרות שיש לנו להשתמש piggyBac לכוון תאי T לאנטיגנים סרטניים 14, יש לנו גם בשימוש piggyBac להוסיף מתג בטיחות מושרה על מנת לחסל תאים מהונדסים גנטיים במידת צורך 7. קיבולת המטען הגדולה של piggyBac גם אפשרה העברת גנים שלמולקולה גדולה rapamycin עמיד mTOR (15 ק&quot;ג) 15. לכן, אנו מציגים מתודולוגיה שאינה נגיפית לגני שינוי יציב של תאי T האנושיים ראשוניים למגוון רחב של מטרות.

סכמטי להדגים את השלבים בשינוי תאי T אנושיים עם גן כתב (eGFP) הגנטי מוצג באיור 1. פלסמידים אלה זמינים על פי בקשה מהמחברים. Schemtic מדגים את השלבים בלימפוציטים מסוג T האנושי מהונדס גנטי עם גן כתב (eGFP) הוא showin באיור 2. זה הכרחי להפעלת תאי T על מנת לקבל אותם להתחלק,...

Watch this Scientific Journal Video about piggyBac Transposon System Modification of Primary Human T Cells at JoVE.com

piggyBac Transposon System Modification of Primary Human T Cells

אנו מתארים שיטה גנטית כדי לשנות את תאי T אנושיים ראשוניים עם transgene שימוש שאינו ויראלי<em&gt; PiggyBac</em&gt; מערכת transposon. תאי T שונים לשימוש<em&gt; PiggyBac</em&gt; Transposon ביטוי transgene מערכת תערוכה יציב.

שינוי מערכת Transposon piggyBac של תאי T אנושיים ראשוניים

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

piggybac-transposon-system-modification-of-primary-human-t-cells

Research

JoVE Journal

Immunology and Infection

piggyBac Transposon System Modification of Primary Human T Cells

17.1K Views.  Baylor College of Medicine. אנו מתארים שיטה גנטית כדי לשנות את תאי T אנושיים ראשוניים עם transgene שימוש שאינו ויראלי PiggyBac מערכת transposon. תאי T שונים לשימוש PiggyBac Transposon ביטוי transgene מערכת תערוכה יציב.

Video: piggyBac Transposon System Modification of Primary Human T Cells

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

הרחבת דם היקפיים האדם γδ T תאים באמצעות Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

דור של מושרה תאים T רגולטוריים של האדם תאים ראשוניים טי נאיביות וזיכרון

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

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 ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/&#955;pir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, &#963;22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.

זיהוי גנים הקשורים לרומן אלגינט ייצור ב<em&gt; Pseudomonas aeruginosa</em&gt; שימוש מיני<em&gt; Himar1</em&gt; Mutagenesis תיווך transposon מרינר

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

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 ...

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific subpopulations.

הערכה של מאפייני המערכת החיסוניים של תאי גזע mesenchymal האדם (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

הערכה של הממשק סינפטית של תאי T האנושי העיקרי של דם היקפיים ורקמות הלימפה

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

זיהום פנאומטי של תאים אנושיים ראשוניים של האדם בזרימה קבועה

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

ייצור תאי CAR-T מהונדסים על-ידי CRISPR האנושי

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with ...

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.