עבודה זו נתמכה על ידי NIH R01 CA104757 גרנט (MH Manjili). בהכרת תודה אנו מכירים את התמיכה של VCU מאסי לחקר הסרטן וקרן העמים לחקר הסרטן.

1. בידוד של לימפוציטים 5 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בודד הגידול ניקוז בלוטות הלימפה או spleens מן הגידול נושאות FVBN202 העכברים הטרנסגניים ולהכין ההשעיה תא יחיד קר כקרח RPMI1640 בתוספת 10% FBS. B / אני ההפעלה של 50 מ&quot;ל פוליפרופילן תוצאות צינורות חרוטי בתשואה טי תא גדול לעומת צינורות קלקר. קטמין ו Xylazine מוזרקים ip עבור הרדמה. פריקה צוואר הרחם משמש כשיטה של ​​המתת חסד. </li><li style=";text-align:right;direction:rtl"> התרבות התאים (10 6 תאים / מ&quot;ל) ב בינוני מלא המכיל 15% FBS עם bryostatin-1 (5 ננומטר) ionomycin (1 מיקרומטר) יחד עם 80 U / mL של IL-2 (Peprotech) עבור 16 ח </li><li style=";text-align:right;direction:rtl"> לשטוף את התאים שלוש פעמים עם המדיום חמים (37 מעלות צלזיוס) והתרבות 10 6 תאים / מ&quot;ל במדיום להשלים עם IL-7 (10 ng / mL) ו-IL-15 (10 ng / mL) (Peprotech) עבור 24 שעות . </li><li style=";text-align:right;direction:rtl"> דופק את התאים עם IL-2 (40 U / mL) במשך 24 ח </li><li style=";text-align:right;direction:rtl"> פיצול תאים והתרבות אותם עם IL-7 ו - IL-15 (10 ng / mL) במשך 4 ימים נוספים. שנה בינונית לפצל את התאים אם יש צורך כל 2 ימים. </li></ol> 2. קביעת הרחבת מקפלים של תאים T על ידי ספירת תאים cytometry זרימה ניתוח 5 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Cell נחשב על ידי מיקרוסקופ אור <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן דילול התא המתאים (1:100) ב trypan כחול ולהוסיף כמה μL על hemocytometer </li><li style=";text-align:right;direction:rtl"> הרוזן 9 ריבועים לקבוע מספר תאים על ידי חלוקת סך ספירת תא למספר תאים כפול פקטור התימהול. התוצאות יציג מספר x 10 4 תאים / מ&quot;ל. </li></ol></li><li style=";text-align:right;direction:rtl"> קביעת שיעור CD8 + CD4 + ו ותאי T בתאי מורחבת על ידי זרימת cytometry <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בלוק הלא ספציפית מחייב של נוגדנים לקולטנים על ידי Fc culturing התאים עם הנוגדנים anti-CD16/CD32 (Biolegend) במשך 20 דקות על הקרח ולאחר מכן לשטוף את התאים פעמיים עם 2 מ&quot;ל של קר כקרח PBS בתוספת אזיד הנתרן 1% . </li><li style=";text-align:right;direction:rtl"> הכתם התאים על ידי culturing עם FITC-CD4 ו PE-CD8 נוגדנים עבור 20 דקות על הקרח ולאחר מכן לשטוף את התאים פעמיים עם 2 מ&quot;ל של קר כקרח PBS בתוספת 1% ו FBS אזיד הנתרן 0.1%. </li><li style=";text-align:right;direction:rtl"> תקן את התאים paraformaldehyde 1% ולהפעיל דגימות על Beckman Coulter FC 500 ולנתח בעזרת גרסה 4.3 Summit תוכנה. </li></ol></li></ol> 3. קביעת גידולים הספציפיות של Vivo לשעבר מורחבת תאים T <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תרבות vivo לשעבר מורחבת הלימפוציטים בינוני מלא ביחס 10:01 עם מוקרן Neu תאים סרטניים חיובי MMC (15,000 rad) עבור 24 ח 5 </li><li style=";text-align:right;direction:rtl"> קציר supernatants ולאחסן ב -80 ° C עד בשימוש. 5,6 </li><li style=";text-align:right;direction:rtl"> זיהוי IFN-γ באמצעות עכבר IFN-γ ELISA הגדר (BD Pharmingen) על פי פרוטוקול של היצרן. 5,6 </li></ol> 4. קביעה אנטי סרטניים פונקציה של Vivo תאים לשעבר מורחבת T 5,6 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דגירה T תאים עם תאים סרטניים 10:01 מפעיל: יחסי יעד 48 שעות להשלים בינוני בבית בינוני 3 מ&quot;ל מלאה (RPMI-1640 בתוספת 100U / mL של פניצילין, 100μg / סטרפטומיצין מ&quot;ל, 10% FBS, גלוטמין ו - β - mercaptoethanol) ו 20U / mL של IL-2 (Peprotech) ב 6 מנות התרבות גם CO 37 ° C / 5% 2. </li><li style=";text-align:right;direction:rtl"> לבצע שלוש נוגדן צבע מכתים עבור Neu (anti-c-Erb2/c-neu, לשבט-4, Calbiochem) ואחריו PE-אנטי עכבר IgG, Annexin V-FITC ו יודיד Propidium (PI) על פי פרוטוקול של היצרן (BD Pharmingen) </li><li style=";text-align:right;direction:rtl"> שער על תאים סרטניים Neu חיובי וניתוח כדאיות (Annexin V-/PI-) של תאים סרטניים </li></ol> 5. עכבר דגם של סרטן השד FVBN202 נקבות העכברים הטרנסגניים (Charles River Laboratories) יכול לשמש מקור הגידול בתאי T-reactive. עכברים אלו overexpress עכברוש לא ממושכת Neu transgene לפי תקנה של האמרגן MMTV וכתוצאה מכך לפתח סרטן השד ספונטנית בין 4-10 חודשים של גיל 7. עכברים אלו לפתח היפרפלזיה החלב טרום ממאיר דומה קרצינומה ductal באתרם (DCIS) לפני התפתחות carcinoma8 ספונטנית. ספונטנית נושאות גידולים בעכברים המשמשים התורמים של תאים T. 6. נציג תוצאות: הפעלה של תאים עם T / B אני במשך 16 שעות התוצאות בהרג של תמימים בתאי T, כי הם לא רגישים עם הגידול in vivo. לאחר סלקטיביות B / אני הגידול ותאי T-reactive הם להרחיב עד 2.8 פי בתרבות 6 ימים עם שרשרת גאמא ציטוקינים (איור 1). שני CD8 + CD4 + ו ותאי T מורחבות באופן שווה עם שרשרת גאמא ציטוקינים (איור 2). Ex-vivo מורחבת בתאי T להראות היענות גבוהה נגד גידולים בעכברים כי התורם היו רגישים, כפי מוערך על ידי ייצור של IFN-γ בנוכחות קרצינומה של העכבר Neu חיובי החלב (MMC) תאים סרטניים (איור 3). Vivo לשעבר מורחבת בתאי T יכול לגרום אפופטוזיס של cel חיובי הגידול MMC Neuls כדאיות כזו של תאים סרטניים טיפות של% 92 עד% 61 בתוך 48 שעות (איור 4). <img src="/files/ftp_upload/2381/2381fig1.jpg" alt="חסר תרשים" /> באיור 1. התרחבות מקפלים של לימפוציטים בנקודות זמן שונות לאחר הפעלת B / אני (יום 1) והרחבת vivo לשעבר עם ציטוקינים שרשרת גאמא (יום 3, 5 ו 7) <img src="/files/ftp_upload/2381/2381fig2.jpg" alt="חסר תרשים" /> איור 2. האחוז הכולל של CD4 + ו - CD8 + T תאים לפני ואחרי הרחבת 7 ימים עם ציטוקינים שרשרת גאמא. <img src="/files/ftp_upload/2381/2381fig3.jpg" alt="חסר תרשים" /> איור 3. גידולים מגורה IFN-γ הייצור על ידי בתאי T שבודדו נושאות גידולים בעכברים לפני ואחרי הרחבת 7 ימים עם ציטוקינים שרשרת גאמא, באמצעות IFN-γ ELISA <img src="/files/ftp_upload/2381/2381fig4.jpg" alt="חסר תרשים" /> איור 4. לתפקד ציטוטוקסיות של vivo לשעבר מורחבת בתאי T עם שרשרת גאמא ציטוקינים נגד קרצינומה של העכבר Neu חיובי החלב (MMC) תאים סרטניים

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אין ניגודי אינטרסים הכריז.

הרחבת סלקטיבי של גידולים בתאי T-reactive עם פונקציה אנטי הגידול מפעיל יכולה להיות מושגת על ידי שימוש בפרוטוקול המוצע B / אני הפעלה והרחבה vivo לשעבר עם שרשרת גאמא ציטוקינים IL-2, IL-7 ו IL-15. בעוד IL-2 הוא תא T גורם הצמיחה שיכול לתמוך הבידול וההתרחבות של אנטיגן ספציפי בתאי T, IL-7 יכול לעכב אפופטוזיס של תאים T ותמיכה הכדאיות שלהם במהלך התרחבות. IL-15 T יכול לתמוך בזיכרון תאים חשובים להפקת לטווח ארוך אנטי הגידול התגובות על טיפול המאמצת תא T 9-11. שינוי הסדר ועל שילוב של ציטוקינים אלה יכולים להשפיע על התמיינות של תאים T מורחבת אשר בתורו עשוי לשפר או להפחית 12 אנטי הגידול שלהם יעילות. הפרוטוקול המוצע לא ידרוש זיהוי של אנטיגנים סרטניים. הרחבת סלקטיבי של הגידול T-reactive תוצאות תאים בייצור של מספרים גבוהים של אנטי הגידול ותאי T, שניתן להשתמש בהם לטיפול המאמצת תא T של חולי סרטן. הראינו בעבר כי vivo לשעבר מורחבת בתאי T בעלי חיים מוגנים מפני סרטן השד לאחר טיפול המאמצת תא T.

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Bryostatin 1</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B7431-10ug</td>
<td>&nbsp;</td>
<tr>
<td>Ionomycin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>407950</td>
<td>&nbsp;</td>
<tr>
<td>Mouse IL-7</td>
<td>&nbsp;</td>
<td>PeproTech</td>
<td>217-17</td>
<td>&nbsp;</td>
<tr>
<td>Mouse IL-15</td>
<td>&nbsp;</td>
<td>PeproTech</td>
<td>210-15</td>
<td>&nbsp;</td>
<tr>
<td>Human IL-2</td>
<td>&nbsp;</td>
<td>PeproTech</td>
<td>200-02</td>
<td>&nbsp;</td>
<tr>
<td>RPMI1640</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11875</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Gemini</td>
<td>100-106</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-002-CI</td>
<td>&nbsp;</td>
<tr>
<td>L- glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030081</td>
<td>&nbsp;</td>
<tr>
<td>&beta;- mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M7522</td>
<td>&nbsp;</td>
<tr>
<td>anti-CD16/32 antibody</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>101302</td>
<td>&nbsp;</td>
<tr>
<td>Annexin V-FITC Apoptosis Detection Kit</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>556547</td>
<td>&nbsp;</td>
<tr>
<td>FITC-CD4</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>100406</td>
<td>&nbsp;</td>
<tr>
<td>PE-CD8</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>100708</td>
<td>&nbsp;</td>
<tr>
<td>anti-c-Erb2/c&#x2013;Neu</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>OP16</td>
<td>&nbsp;</td>
<tr>
<td>PE- anti mouse IgG</td>
<td>&nbsp;</td>
<td>Biolegend</td>
<td>405307</td>
<td>&nbsp;</td>
<tr>
<td>formaldehyde</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>04018</td>
<td>&nbsp;</td>
<tr>
<td>Hemocytometer</td>
<td>&nbsp;</td>
<td>Hycor</td>
<td>87144</td>
<td>&nbsp;</td>
<tr>
<td>Light microscope</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>V200073</td>
<td>&nbsp;</td>
<tr>
<td>Mouse IFN-&gamma; ELISA set</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>555138</td>
<td>&nbsp;</td>
<tr>
<td>Cell culture flasks</td>
<td>&nbsp;</td>
<td>Greiner</td>
<td>658175</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

ex vivo expansion of tumor-reactive t cells by means of bryostatin 1/ionomycin and the common gamma chain cytokines formulation

נמסר כי חולי סרטן השד יש קיימים התגובות החיסוניות נגד גידולים 1,2 שלהם. עם זאת, התגובה החיסונית כאלה אינן מספקות הגנה מלאה מפני התפתחות או הישנות סרטן השד. כדי להתגבר על הבעיה על ידי הגדלת תדירות הגידול-reactive בתאי T, חיסוני המאמצת הועסק. מגוון של פרוטוקולי שימשו להרחבת גידול ספציפי בתאי T. פרוטוקולים אלה, לעומת זאת, מוגבלים לשימוש vivo אנטיגנים סרטניים לשעבר להפעלת אנטיגן ספציפי בתאי T. ממש לאחרונה, נפוץ שרשרת גאמא ציטוקינים כגון IL-2, IL-7, IL-15, IL-21 שימשו לבד או בשילוב להגברת אנטי הגידול התגובה החיסונית 3. עם זאת, לא ברור מה ניסוח יעבוד בצורה הטובה ביותר להרחבת הגידול ותאי T-reactive. כאן אנו מציגים פרוטוקול להפעלת סלקטיבית והרחבת הגידול ותאי T-reactive ממודל FVBN202 עכבר מהונדס של סרטן שד חיובי HER-2/neu לשימוש בטיפול T המאמצת התא של סרטן השד. הפרוטוקול כולל הפעלה של תאים T עם bryostatin-1/ionomycin (B / I) IL-2 בהעדר אנטיגנים סרטניים במשך 16 שעות. B / הפעלה אני מחקה אותות תאיים כי התוצאה הפעלת תא T על ידי הגדלת הפעילות חלבון kinase C, סידן תוך תאי, בהתאמה 4. פרוטוקול זה במיוחד הגידול מפעיל ספציפי בתאי T תוך הרג ותאי T לא רלוונטי. B / אני המופעל בתאי T הם מתורבתים עם IL-7 ו - IL-15 במשך 24 שעות ו פעמו אז עם IL-2. לאחר 24 שעות, ותאי T נשטפים, מפוצל, ותרבותיים עם IL-7 + IL-15 במשך 4 ימים נוספים. יעילות הגידול סגוליות ואנטי גידול של ex vivo תאים מורחבת T נקבע.

Watch this Scientific Journal Video about Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation at JoVE.com

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

פרוטוקול יעיל עבור<em&gt; לשעבר vivo</em&gt; התפשטות של הגידול ותאי T-reactive מן הגידול ניקוז בלוטות הלימפה או אחרות ברקמות הלימפה משני נושאות גידולים המארחים מתואר. פרוטוקול זה מרחיב סלקטיבי גידולים ספציפיים בתאי T לשימוש חיסוני המאמצת של סרטן השד.

אקס הרחבה vivo של גידול תאים T-reactive באמצעות 1/Ionomycin Bryostatin ואת גמא נפוצים שרשרת גיבוש ציטוקינים

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

ex-vivo-expansion-tumor-reactive-t-cells-means-bryostatin-1ionomycin

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

14.7K Views.  Virginia Commonwealth University- Massey Cancer Center. פרוטוקול יעיל עבור לשעבר vivo התפשטות של הגידול ותאי T-reactive מן הגידול ניקוז בלוטות הלימפה או אחרות ברקמות הלימפה משני נושאות גידולים המארחים מתואר. פרוטוקול זה מרחיב סלקטיבי גידולים ספציפיים בתאי T לשימוש חיסוני המאמצת של סרטן השד.

Video: Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

HLA-איג בהתבסס Antigen תאים מלאכותיים הצגת עבור יעיל לשעבר vivo הרחבת CTL האדם

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Ex vivo הדמיה של תאים T ב Slices צומת Murine לימפה עם widefield ו מיקרוסקופים confocal

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

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  &#947;&#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 ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

בעקבות Cell-גורל ב<em&gt; א coli</em&gt; לאחר זיהום על ידי phage מבדה

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

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

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

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

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

Antigen המלאכותי הצגת תא הפעלה (aAPC) מתווכות והרחבת תאי T קטלניים טבעיים

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

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

הפרדה<em&gt; Plasmodium falciparum</emאריתרוציטים&gt; שלב מאוחר נגועים באמצעי מגנטי

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

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 ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

<em&gt; Ex vivo</em&gt; דגם של התקפת T-Cell-מכוון oligodendrocyte בפרוסות המוח חריף

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

תאי T ללכוד חיידקים על ידי Transinfection מתאים דנדריטים

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

אקס הרחבה vivo של גידול תאים T-reactive באמצעות 1/Ionomycin Bryostatin ואת גמא נפוצים שרשרת גיבוש ציטוקינים

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HLA-איג בהתבסס Antigen תאים מלאכותיים הצגת עבור יעיל לשעבר vivo הרחבת CTL האדם

Ex vivo הדמיה של תאים T ב Slices צומת Murine לימפה עם widefield ו מיקרוסקופים confocal

הרחבת דם היקפיים האדם γδ T תאים באמצעות Zoledronate

בעקבות Cell-גורל ב

Antigen המלאכותי הצגת תא הפעלה (aAPC) מתווכות והרחבת תאי T קטלניים טבעיים

הפרדה

הערכת חישה המולדת של CD4 הנגוע ב- HIV-1

תאי T ללכוד חיידקים על ידי Transinfection מתאים דנדריטים

ובידוד