Name: generating de novo antigen-specific human t cell receptors by retroviral transduction of centric hemichain
Uploaded: 2016-10-25T15:00:00
Description: Watch this Scientific Journal Video about Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain at JoVE.com

This work was supported by NIH grant R01 CA148673 (NH); the Ontario Institute for Cancer Research Clinical Investigator Award IA-039 (NH); BioCanRX Catalyst Grant (NH); The Princess Margaret Cancer Foundation (MOB, NH); Canadian Institutes of Health Research Canada Graduate Scholarship (TG); Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship (TG); Province of Ontario (TG, MA); and Guglietti Fellowship Award (TO). HLA and CD1d monomers were kindly provided by the NIH tetramer core facility.

1. הכנת retroviral לבנות קידוד TCR Hemichain עניין <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Linearize וקטור PMX לאפשר החדרה של גנים TCR בשלבים הבאים. לעכל את ה- DNA פלסמיד עם אנזימי הגבלה EcoRI ו NotI ב 37 מעלות צלזיוס במשך 3 שעות (טבלה 1) 8. </li><li style=";text-align:right;direction:rtl"> לבצע א?...

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הדרישה הראשונה עבור יישום מוצלח של שיטה זו היא השיגה יעילות תמרה מספקת של תאי T העיקריים עם hemichain עניין. מניסיוננו, השילוב של שימוש PG13 כמו שורת תאי אריזת PMX כתוצאות וקטור retroviral בביטוי יציב ויעיל של הגן הציג בתאי T עיקריים אדם. תאי אריזות PG13 יכולים להיות משובט מתא בודד כד?...

לקולטנים המצויים בתאי T (TCRs) הם קולטנים החיסונית heterodimeric אדפטיבית שהביעו לימפוציטים מסוג T, מורכבת שרשרת TCRα ו TCRβ. הם מופקים באמצעות התארגנות סומטיים של V (D) קטעי גן J, המייצר רפרטואר מגוון מאוד מסוגל לזהות תצורות כמעט בלתי מוגבלות של מתחמי HLA / פפטיד. מבחינה קלינית, תאי T שהונדסו ספציפיים TCRs clonotypic עבור אנטיגנים גידולים הקשורים הוכיחו יעילות במגוון סוגי סרטן 1. עם זאת, רבי TCRs משובט למטרה זו חסרה זיקה מספקת את האנטיגן של עניין, מגבילות מריחות הטיפולית שלהם. כאן, אנו מתארים שיטה להתגבר על מגבלה זו על TCRs הקיים תוך ניצול-מרכזיות שרשרת. זה כבר דווח כי hemichain אחד TCR יכול לשחק תפקיד דומיננטי יותר לאות הוקרה על האנטיגן היעד 2, כאן כינה מרכזית. ניתוחים מבניים קריסטל הראו כי ממוקד אחדhemichain של TCR יכול להסביר את רוב טביעת הרגל על פפטיד MHC / 3,4 המורכב. תוך שימוש בקונספט זה, שהראנו בעבר כי SIG35α TCRα תוכל לשייך את רפרטואר מגוון של שרשראות TCRβ ולתחזק תגובתיות כנגד פפטיד MART1 27-35 שהציג HLA-A2 5. תוצאות דומות התקבלו עם TAK1 TCR, שבו hemichain TCRβ ממוקדת זיווג עם שרשראות TCRα שונים ומתוחזק תגובתיות לייצור הפפטיד WT1 235-243 שהציג HLA-A24 6. שניהם MART1 ו WT1 הם אנטיגנים גידולים הקשורים. שרשרת-מרכזיות יושמה גם ללמוד ההכרה אנטיגן של הרוצח הטבעי משתנה-מוגבל CD1d (iNKT) TCRs, על ידי הצמדת שרשרת Vα24-Jα18 (Vα24i) TCRα משתנה של TCRs iNKT אדם עם 7 שרשראות Vβ11 TCRβ שונים. בכל המקרים, הצלחנו ליצור רפרטואר דה נובו של TCRs על ידי טרנסמחוללות hemichain TCR ממוקדת לתאי T דם היקפיים, שבו hemichain הציג לזווג עם TCRα אנדוגני או נגד רשתות TCRβ. בעיקרו של דבר, hemichain הממוקדת משמשת פיתיון שיכול לשמש לזיהוי נגד השרשרות המתאימות, אשר כאשר לזווג יחד יוצרות TCRs המקיים את הספציפיות אנטיגן של עניין, עדיין משתנות זיקה. ברפרטואר הרומן אלה, הצלחנו לבודד TCRs clonotypic עם כוח אינטראקציה משופרת נגד אנטיגן היעד לעומת TCRs קיימים. לכן, אנו מאמינים בשיטה זו תאיץ את צנרת זיהוי TCRs האופטימלי עבור יישומים קליניים.

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			<td height="44" style="height:44px;width:216px;">0.05% Trypsin-EDTA</td>
			<td style="width:180px;">Wisent Bioproducts</td>
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			<td height="44" style="height:44px;width:216px;">293GPG cells</td>
			<td style="width:180px;">Generated by Ory et al. (ref 8)</td>
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			<td height="44" style="height:44px;width:216px;">Agar</td>
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			<td height="44" style="height:44px;width:216px;">Ampicillin sodium salt</td>
			<td style="width:180px;">Wisent Bioproducts</td>
			<td style="width:139px;">400-110-IG</td>
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			<td height="44" style="height:44px;width:216px;">Chloroform</td>
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			<td height="44" style="height:44px;width:216px;">Deoxynucleotide (dNTP) Solution Mix</td>
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			<td height="44" style="height:44px;width:216px;">DMEM, high glucose, pyruvate</td>
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			<td height="44" style="height:44px;width:216px;">Fetal Bovine Serum</td>
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			<td height="44" style="height:44px;width:216px;">Filter</td>
			<td style="width:180px;">Corning</td>
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			<td>0.45 mm pore SFCA membrane</td>
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			<td style="width:180px;">Biolegend</td>
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			<td>clone ME20.4</td>
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			<td height="44" style="height:44px;width:216px;">FITC-conjugated anti-human CD3 mAb</td>
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			<td>clone UCHT1</td>
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			<td height="44" style="height:44px;width:216px;">Gentamicin</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:139px;">15750078</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Gibson Assembly Master Mix</td>
			<td style="width:180px;">New England Biolabs</td>
			<td style="width:139px;">E2611L</td>
			<td>used for multi-piece DNA assembly</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">HLA-A2 pentamer</td>
			<td style="width:180px;">Proimmune</td>
			<td style="width:139px;">depends on antigenic peptide</td>
			<td>HLA-A2/MART1 multimer used here was purchased from Proimmune</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">HLA/CD1d monomers</td>
			<td style="width:180px;">NIH Tetramer Core Facility</td>
			<td style="width:139px;"></td>
			<td>multimerize monomers according to protocol provided by NIH tetramer core facility</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Human AB serum</td>
			<td style="width:180px;">Valley Biomedical</td>
			<td style="width:139px;">HP1022</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">human CD3 microbeads</td>
			<td style="width:180px;">Miltenyi Biotec</td>
			<td style="width:139px;">130-050-101</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">IOTest Beta Mark TCR V beta Repertoire Kit</td>
			<td style="width:180px;">Beckman Coulter</td>
			<td style="width:139px;">IM3497</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Jurkat 76 cells</td>
			<td style="width:180px;">Generated by Heemskerk et al. (ref 10)</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">LB Broth</td>
			<td style="width:180px;">Wisent Bioproducts</td>
			<td style="width:139px;">800-060-LG</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">LS MACS column</td>
			<td style="width:180px;">Miltenyi Biotec</td>
			<td style="width:139px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">NEB 5-alpha Competent E. coli</td>
			<td style="width:180px;">New England Biolabs</td>
			<td style="width:139px;">C2987I</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">NEBuffer 3.1</td>
			<td style="width:180px;">New England Biolabs</td>
			<td style="width:139px;">B7203S</td>
			<td>used for EcoRI and NotI digestion</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">NotI</td>
			<td style="width:180px;">New England Biolabs</td>
			<td style="width:139px;">R0189S</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">NucleoBond Xtra Midi</td>
			<td style="width:180px;">Macherey-Nagel</td>
			<td style="width:139px;">740410</td>
			<td>used for plasmid purification</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">PC5-conjugated anti-human CD8 mAb</td>
			<td style="width:180px;">Beckman Coulter</td>
			<td style="width:139px;">B21205</td>
			<td>clone B9.11</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">PG13 cells</td>
			<td style="width:180px;">ATCC</td>
			<td style="width:139px;">CRL-10686</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Phusion HF Buffer Pack</td>
			<td style="width:180px;">New England Biolabs</td>
			<td style="width:139px;">B0518S</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Phusion&#160;High-Fidelity DNA Polymerase</td>
			<td style="width:180px;">New England Biolabs</td>
			<td style="width:139px;">M0530L</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">pMX retroviral vector</td>
			<td style="width:180px;">Cell Biolabs</td>
			<td style="width:139px;">RTV-010</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">polybrene</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:139px;">H-9268</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Proleukin (recombinant human interleukin-2)</td>
			<td style="width:180px;">Novartis</td>
			<td style="width:139px;">by Rx only</td>
			<td>equivalent product can be purchased from Sigma-Aldrich</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Purified anti-human CD3 antibody</td>
			<td style="width:180px;">Biolegend</td>
			<td style="width:139px;">317301</td>
			<td>clone OKT3, used for T cell stimulation</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">RPMI 1640</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:139px;">11875119</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">SA-PE</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:139px;">S866</td>
			<td>used for multimerizing monomers from NIH tetramer core facility</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">SMARTer RACE 5&#39;/3&#39;&#160; Kit</td>
			<td style="width:180px;">Clontech</td>
			<td style="width:139px;">634858</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Sterile water</td>
			<td style="width:180px;">Wisent Bioproducts</td>
			<td style="width:139px;">809-115-LL</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">SuperScript III First-Strand Synthesis System</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:139px;">18080051</td>
			<td>for cDNA synthesis</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Syringe</td>
			<td style="width:180px;">BD</td>
			<td style="width:139px;">301604</td>
			<td>10 mL, slip tip</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Tetracycline hydrochloride</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:139px;">T7660</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">TransIT-293</td>
			<td style="width:180px;">Mirus Bio</td>
			<td style="width:139px;">MIR 2700</td>
			<td>used to transfect 293GPG cells</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">TRIzol Reagent</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:139px;">15596026</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating de novo antigen-specific human t cell receptors by retroviral transduction of centric hemichain

לקולטנים מצויים בתאי T (TCRs) משמשים קליני לכוון את הספציפיות של תאי T למקד גידולים כמו אופנות מבטיחות של אימונותרפיה. לכן, שיבוט TCRs הספציפית אנטיגנים סרטניים הקשורים שונים הייתה המטרה של מחקרים רבים. כדי לעורר תגובה של תאי T אפקטיבי, TCR חייב להכיר אנטיגן היעד עם זיקה אופטימלית. עם זאת, שיבוט כזה TCRs כבר אתגר ורבים זמין TCRs להחזיק זיקה תת אופטימלית עבור אנטיגן מאותו מקור. בפרוטוקול זה, אנו מתארים שיטה של שיבוט דה נובו זיקה גבוהה TCRs אנטיגן ספציפי באמצעות TCRs הקיים תוך ניצול מרכזיות hemichain. זה ידוע כי עבור חלק TCRs, כל TCRα או hemichain TCRβ לא לתרום באופן שווה הכרת אנטיגן, ואת hemichain הדומיננטי הוא המכונה hemichain הממוקדת. הראינו כי על ידי התאמת hemichain הממוקדת עם שרשרות דלפק שונות מדלפק השרשרת המקורית, אנו מסוגלים לשמור על אנטיגן specificity, תוך ויסות כוח האינטראקציה שלה עבור אנטיגן מאותו מקור. לפיכך, את הפוטנציאל הטיפולי של TCR נתון ניתן לשפר על ידי אופטימיזציה של זיווג בין hemichains ממוקדת הדלפק.

ללא ידע מוקדם של אשר hemichain הוא שרשרת ממוקדת, שרשרת TCRα ו TCRβ צריך להיות משובטים בנפרד transduced לתאי T בדם ההיקפי, אשר נעשה במקרה של HLA-A24 / WT1 תגובתי TAK1 TCR (איור 1). תמרה של TAK1β ניב תדירות ניכר גבוהה של תאי T אנטיגן ספציפי. לעומת זאת, תמרה של hemichain הלא ממו?...

Watch this Scientific Journal Video about Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain at JoVE.com

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

בזאת אנו מתארים שיטה חדשנית להפקה לקולטנים המצויים בתאי T אנטיגן ספציפי (TCRs) על ידי התאמת TCRα או TCRβ של TCR קיים, שהוא בעל אנטיגן-הסגולי של עניין, עם hemichain המשלים של רפרטואר קולטן תא ההיקפי T. TCRs שנוצר דה נובו לשמר אנטיגן-סגולי עם משתנה זיקה.

יצירה<em&gt; דה נובו</em&gt; לקולטנים המצויים בתאי אנטיגן ספציפי האדם T על ידי retroviral התמרה של Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

The overall goal of this experimental procedure is to generate De Novo T Cell Receptors or TCRS, recognizing a specific antigen of interest. This method can help isolate optimal T Cell Receptors in a field of gene engineered immunotherapy. The main advantage of this technique is only a single alpha or beta hemi-chain needs to be isolated to generate new entrance specific T Cell Receptors.After eluting the TCR, PCR product clone the gene by combining each linear fragment with a commercially available assembly master mixery agent and incubating the mixture at 50 degrees Celsius for one hour. At the end of the incubation, transform chemically confident E Coli with the assembled plasmin following the manufacturers protocol and seed the transformed bacteria onto agar plates for 18 hour incubation at 37 degrees Celsius. The next day, transfer a single bacterial colony into 50 milliliters of lysogeny broth medium for 16 hours in a shaking incubator at 37 degrees celsius and 200 revolutions per minute.Then, using a commercially available Midiprep kit, according to the manufacturer's protocol, purify the plasmin and dilute the DNA to a concentration of around one microgram per microliter for the 293GPG cell transfection. One day after the transfection, replace the transfection medium from the 293GPG cell culture with fresh DMEM medium. Two days after changing the medium, collect the culture medium in a syringe, followed by it's passage through a 0.45 micron filter to collect the virus.Next seed one times ten to the fifth PG-13 cells in a T-25 flask overnight. The next day aspirate the culture medium and add 1.5 milliliters of the 293GPG derived virus, 1.5 milliliters of DMEM medium, and eight micrograms per milliliter of Polygrain. Change the medium once a day for four days to establish a stable PG-13 packaging cell line for producing a retro virus, encoding a TCR hemi-chain of interest.24 hours after the last infection, replace the culture medium with fresh DMEM medium. To harvest the virus from the transduced cell line, culture two times ten to the sixth PG-13 packaging cells in a T-75 flask with 10 milliliters of fresh DMEM medium. And collect the virus three days after seeding, as just demonstrated.For T cell activation, culture two times ten to the seventh PBMCs per well in a six well plate in five milliliters of RPMI medium supplemented with Recombinant Human IL-2 and anti-CD3 monoclonal antibody. Three days post stimulation, harvest the activated T Cells for centrifugation and resuspend 0.5 to one times ten to the six cells in one milliliter of PG-13 virus and one milliliter of RPMI medium. Supplement it with Recombinant Human IL-2 per well, proceeding in a 24 well plate.When all of the cells have been deposited, centrifuge the plate and transfer the plate into a cell cultured incubator. After 24 hours harvest the cells for centrifugation. Then, resuspend the T Cells pellet in one milliliter of PG-13 virus and one milliliter of RPMI by medium supplemented with IL-2 and centrifuge the plate as just demonstrated.24 hours after the last infection, harvest the cells for centrifugation and resuspend the T Cells in RPMI medium supplemented with IL-2 alone. Return the cells to the incubator for two to three more days of culture. Then stain the cells with a HLA multimer for 30 minutes at four degrees Celsius.Followed by anti-human CD3 and clone receptor monoclonal antibodies at four degrees Celsius for 15 minutes. Sort the multimer positive cells by flow cytometry using the appropriate controls. Seed five times ten to the fourth Jurkat cells per well in a 24 well plate in one milliliter of RPMI medium and one milliliter of 293GPG virus per Jurkat cell transfection with the centric TCR hemi-chain of interest.Centrifuge the plate, and place the plate in the cell culture incubator. After 24 hours collect the cells for centrifugation and resuspend the hemi-chain transduced Jurkat 76 cell pellet in fresh RPMI medium for further culture. To fully reconstitute the T Cell Receptor, produce 293GPG virus encoding a TCR counter-chain, cloned from the sorted multimer positive cells.Enter the centric hemi-chain expressing Jurkat 76 cells as just demonstrated. Three to five days post CD3 purification, stain the clonal tipic TCR expressing transfectants with the appropriate HLA multimer and cell surface staining antibodies of interest. And analyze the cells by flow cytometry to verify the antigen specificity.Without prior knowledge of which hemi-chain is chain centric, The TCR alpha and TCR beta chains should be separately clone and transduced to peripheral blood T-cells. As for this HLA A-24 WT1 reactivate TAK1 T Cell Receptor. TAK1 Beta transsection yields a noticeably higher frequency of antigen specific T Cell.While the transduction of a non centric hemi-chain does not yield to know well positive T Cells as observed for the TAK1 alpha chain. Conversely a single centric hemi-chain can be transduced if it is known to be dominant. Further, T Cells specific for CD1d increase in frequency upon transduction of the centric TCR V alpha 24 hemi-chain.Together these data demonstrate that the introduction of a centric TCR alpha or beta hemi-chain into peripheral blood T cells can generate De Novo TCRs with the antigen specificity of interest. Moreover De Novo TCR alpha counter-chains, paired with TAK1 beta, stained with either a lower or higher intensity by the WT1 antigen complex than the parental TAK1 alpha beta pairing. Similarly, SAK-35 alpha paired with unique counter-chains, recognizes HLA A2 MART1 with a moderate to high avidity.While attempting this procedure to obtain the maximal transection efficiency, it is important to maintain primary T Cells under the optimal culture conditions. Following this procedure, novel HRE class 2 restricted entrance specific T Cell Receptors can be cloned by exploiting the chain centricity. After watching this video you should have a good understanding of how to isolate and reconstitute T Cell Receptor genes by molecular cloning and retroviral transduction.Don't forget that working with replication defective retrovirus can potentially be hazardous and that precaution such as wearing gloves and protective garments should always be taken while performing this procedure.

Research

JoVE Journal

Immunology and Infection

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Retroviral Transduction of T-cell Receptors in Mouse T-cells

התמרה retroviral של T-cell קולטנים עכבר מתאי T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

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 ...

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

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

תאי מציגי הרוצח המלאכותי Antigen (KaAPC) ליעיל<em&gt; במבחנה</em&gt; דלדול של תאי T אנושי אנטיגן ספציפי

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

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

טכניקות הדמיה כל-החיה והזרימה Cytometric לתגובות ניתוח של אנטיגן ספציפי CD8 + T תא לאחר Nanoparticle חיסון

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

Retroviral התמרה של ובתאים מח עצם כדי ליצור תאי T קולטן Retrogenic עכברים

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Retroviral תמרה של Helper T תאים כגישה גנטית לחקר מנגנוני בקרת הבידול והתפקוד שלהם

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

פיתוח תאי גזע שמקורם אנטיגן ספציפי תקינים בתאי T נגד אוטואימוניות

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

אין ויוו Assay גילוי של הפונקציה Cytolytic תא T אנטיגן ספציפי באמצעות מודל החיסון

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

דור שמסווגת נוגדנים חד-שבטיים אנושי מתאי B אנטיגן ספציפי נדיר במחזור הדם

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

עיבוד פרוטוקול עבור הניתוח של פעפוע וגודל האשכול של קולטני ממברנה על-ידי קרינה פלואורסצנטית מיקרוסקופ תמונה

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using ...