We thank Joon Kim for expert flow cytometry assistance. Research in the authors&#8217; labs is supported by the SCORE program of the National Institutes of Health (grant SC1-GM095402 to B.D.O) and the Canadian Institutes of Health Research (to J.C.Z.P.). J.C.Z.P. is supported by a Canada Research Chair in Developmental Immunology. The biomedical research infrastructure of Hunter College is supported in part by the NIH Research Centers in Minority Institutions (RCMI) program via grant MD007599. We also acknowledge the New York State Stem Cell Science Program (NYSTEM) for its support of the initiation of stem cell research at Hunter College via grant C023048.

<ol>
	<li>Schmitt, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+T+cell+development+and+establishment+of+T+cell+competence+from+embryonic+stem+cells+differentiated+in+vitro.">Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro.</a> Nat Immunol. 5, 410-417 (2004).</li><li>Pui, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1+expression+in+early+lymphopoiesis+influences+B+versus+T+lineage+determination.">Notch1 expression in early lymphopoiesis influences B versus T lineage determination.</a> Immunity. 11, 299-308 (1999).</li><li>Nakano, T., Kodama, H., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+lymphohematopoietic+cells+from+embryonic+stem+cells+in+culture.">Generation of lymphohematopoietic cells from embryonic stem cells in culture.</a> Science. 265, 1098-1101 (1994).</li><li>Schmitt, T. M., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+T+cell+development+from+hematopoietic+progenitor+cells+by+delta-like-1+in+vitro.">Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro.</a> Immunity. 17, 749-756 (2002).</li><li>Chen, J., Lansford, R., Stewart, V., Young, F., Alt, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAG-2-deficient+blastocyst+complementation:+an+assay+of+gene+function+in+lymphocyte+development.">RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development.</a> Proceedings of the National Academy of Sciences of the United States of America. 90, 4528-4532 (1993).</li><li>de Pooter, R. F., Cho, S. K., Carlyle, J. R., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+generation+of+T+lymphocytes+from+embryonic+stem+cell-derived+prehematopoietic+progenitors.">In vitro generation of T lymphocytes from embryonic stem cell-derived prehematopoietic progenitors.</a> Blood. 102, 1649-1653 (2003).</li><li>Holmes, R., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+OP9-DL1+system:+generation+of+T-lymphocytes+from+embryonic+or+hematopoietic+stem+cells+in+vitro.">The OP9-DL1 system: generation of T-lymphocytes from embryonic or hematopoietic stem cells in vitro.</a> Cold Spring Harb Protoc. 2009, (2009).</li><li>Arsov, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+autophagic+protein+beclin+1+early+in+lymphocyte+development.">A role for autophagic protein beclin 1 early in lymphocyte development.</a> J Immunol. 186, 2201-2209 (2011).</li><li>Lahiji, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+TCR-alpha+gene+locus+control+region+activity+in+T+cells+derived+in+vitro+from+embryonic+stem+cells.">Complete TCR-alpha gene locus control region activity in T cells derived in vitro from embryonic stem cells.</a> J Immunol. 191, 472-479 (2013).</li><li>Hirashima, M., Kataoka, H., Nishikawa, S., Matsuyoshi, N., Nishikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+embryonic+stem+cells+into+endothelial+cells+in+an+in+vitro+model+of+vasculogenesis.">Maturation of embryonic stem cells into endothelial cells in an in vitro model of vasculogenesis.</a> Blood. 93, 1253-1263 (1999).</li><li>Nishikawa, S. I., Nishikawa, S., Hirashima, M., Matsuyoshi, N., Kodama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+lineage+analysis+by+cell+sorting+and+culture+identifies+FLK1+VE-cadherin++cells+at+a+diverging+point+of+endothelial+and+hemopoietic+lineages.">Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages.</a> Development. 125, 1747-1757 (1998).</li><li>de Pooter, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+requires+GATA-2+to+inhibit+myelopoiesis+from+embryonic+stem+cells+and+primary+hemopoietic+progenitors.">Notch signaling requires GATA-2 to inhibit myelopoiesis from embryonic stem cells and primary hemopoietic progenitors.</a> J Immunol. 176, 5267-5275 (2006).</li><li>Watarai, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+NKT+cells+in+vitro+from+embryonic+stem+cells+bearing+rearranged+invariant+Valpha14-Jalpha18+TCRalpha.">Generation of functional NKT cells in vitro from embryonic stem cells bearing rearranged invariant Valpha14-Jalpha18 TCRalpha.</a> 115, 230-237 (2010).</li><li>Pipkin, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+transfer+activates+and+delineates+a+locus+control+region+for+perforin.">Chromosome transfer activates and delineates a locus control region for perforin.</a> Immunity. 26, 29-41 (2007).</li></ol>

The authors have nothing to disclose.

מערכת שיתוף תרבות OP9-DL1 נוצלה כדי ללמוד את התפקיד של מוצרי גנים שונים במהלך הפיתוח של סוגי תאי דם מתאי גזע. 8,12,13 זה הוכח גם מודל יעיל כדי ללמוד את הפונקציה של DNA הרגולציה הגן במהלך התמיינות תאים. 9,14 שימוש בגישה זו כחלופה למודלים של עכברים כל יכול להניב חיסכון משמעותי בזמן ועלויות של ניסויים העוסקים בשאלות רבות בסיסיות בhematopoiesis. עם זאת, עיבוד של פרוטוקול זה למטרות אלה מחייב ידיעה של השתנות פוטנציאל בין השיבוטים המסקלין שישמשו בהליכים אלה. ציר הזמן של הפרוטוקול שפורסם הוא expectable מהממוצע, מתוחזק היטב שורת המסקלין לא מניפולציות,. 7 בנוסף, השיבוטים המסקלין נבחרו לדור של עכברי chimeric בדרך כלל הם באיכות גבוהה יותר ויבצע מאוד וחסונה בשיתוף תרבות OP9 . מצד השני, שיבוטים המסקלין מתעוררים ישירות מיציבtransfection עם transgene המשולב ectopically יציג דרגות שונות של יעילות בידול ראשונית הנעה בין ממוצע לממוצע מתחת. הפרוטוקול המתואר כאן מספק לעיכוב 1-2 יום אסטרטגי של שלב 5 מעבר היום כדי לאזן את הבדלים פוטנציאליים אלה לפני הגיוס של hematopoiesis במבחנה. מעבר יום 8 הוא הצעד שבו הביצוע של פרוטוקול זה יש רוב הפוטנציאל לטעויות. סבלנות, תרגול והתבוננות זהירה תאפשר אחד כדי לפתח את הטכניקה אשר מייעלת קציר HPC תוך מזעור ההפרעה monolayer OP9. מעבר ליום 5 ויום 8 שלבים העיקריים, הפרמטרים הקריטיים כרוכים תחזוקה קפדנית תרבית תאים (תאים במיוחד OP9, כפי שתואר לעיל) ותשומת לב מיוחדת לחומרים הכימיים המשמשים בפרוטוקול. מגיב הקריטי ביותר הוא FBS. הרבה מובהק של FBS ישתנו במידה ניכרת ביכולתם לתמוך בהליך זה. הרבה מרובים חייבים להיבדק בoבצו כדי לקבוע אילו יניב בידול חזק בassay זה. מערכת שיתוף התרבות זו יש מגבלות משלה. לדוגמא, מודל זה אינו תומך בהתמיינות של תאי CD4 חיוביים יחידים. אחת סיבות לכך היא חוסר הביטוי של מתחם histocompatibility הגדול תשתית מצגת אנטיגן (MHC) Class II בתאי OP9. מצגת משטח נייד 1 של אנטיגנים על ידי MHC Class II נדרשת להתפתחות תקינה של תאי CD4 SP. תאי CD8 SP שעושים לצאת מייצגים שילוב של שלבי thymocyte CD8 SP בוגרים ולא בשלה. 1 יתר על כן, השתלה מוצלחת של המסקלין נגזרת אבות תא T לעכבר דורש מעבר לפני דרך תרבות איבר הרתי עוברית. 1 עם זאת, טכנולוגיה זו הוכיחה אותה מבטיח כגישת מחקר רב עוצמה לשתי השאלות התאיות ומולקולריות בהתפתחות מערכת חיסונית בדם ושהיו אפשריים בעבר רק כדי לחקור בנאיבו. כך, מלבד מתן דרך רומן להתקדם יותר מהירים על שאלות אלה, אימוץ רחב יותר של מערכת שיתוף תרבות OP9-ESC תהיה ההשפעה הרחבה יותר של הפחתת השימוש בחיות ניסוי.

A cell culture system has been established in which mouse embryonic stem cells (mESC) are differentiated to T cells in vitro.1 This system exploits the ability of Notch signaling to drive T cell differentiation.2 The OP9-DL1 cell line was created by transducing bone marrow-derived OP9 cells3 with a Notch ligand, Delta-like 1 (DL1).4 Activation of the Notch signaling cascade in vitro facilitates T cell development to the exclusion of other cell lineages. With the inclusion of appropriate cytokines, this system provides a cell culture &#8220;microenvironment&#8221; that supports the sequential advancement of mESC toward hematopoietic and ultimately T cell lineages. This system supports the flow cytometric identification of T cells at the various developmental stages seen during normal T cell ontogeny in the thymus. For investigating selected questions relating to T cell development, this procedure has become an attractive alternative to in vivo whole mouse models5 and in vitro fetal thymic organ culture methods used to elicit T cell development from mouse embryonic stem cell derived hematopoietic precursors.6 The major advantage of the OP9 co-culture system is that it involves standard and straightforward cell culture techniques and does not depend on the continual use of experimental animals. 
We follow a detailed, previously published protocol in our experiments using this approach.7 We have utilized this technology to examine the hematopoietic differentiation products of non-manipulated mESC clones, high quality mESC clones handpicked to make chimeric embryos8 and stably-transfected ESC clones coming directly out of drug selection.9 We have noted that the temporal kinetics of initial in vitro differentiation from mESC to mesoderm-like colonies in this model can be variable among individual clones. The mESC-OP9 co-cultures can be visually assessed for progression to mesoderm. While this will usually be completed by the fifth day of co-culture, among individual clones, completion can be delayed for one or two days. Quantitative (~80-90%) mesoderm formation must be achieved prior to transfer in order to obtain optimal hematopoietic progenitor cell (HPC) formation and robust lymphopoiesis. Thus, when working with multiple mESC clones, this &#8220;day 5&#8221; passage is best delayed until all clones complete the transition to mesoderm-like colonies. This enables synchrony of subsequent development among the clones after their transfer into hematopoietic differentiation conditions. Three days after the passaging of the 80-90% mesodermal formations, HPCs are collected from the OP9 monolayers. HPCs can be seeded on new OP9 cells to allow differentiation of monocytic, erythroid and B cell lineages. Alternatively, HPCs can be seeded on OP9-DL1 cells and driven towards T cell development. All in vitro differentiation cultures are provided Flt-3L beginning at day 5, with further addition of IL-7 beginning at day 8. Flow cytometry analyses performed at various time points during the experiment enable monitoring of progress through the stages and lineages of hematopoietic differentiation and T cell development. CD4/CD8 double positive (DP) T cells begin emerging by day 16 of the co-culture, and both DP and CD8 single positive (SP) cells are abundant by day 20. The general outcome and robustness of co-culture is greatly dependent on the ability to visually ascertain the completion of the significant developmental turning points that occur. This protocol aims to be a guide to the recognition of these milestones, as well as the other critical parameters, that are key to successful differentiation.

<table border="0" cellpadding="0" cellspacing="0" width="1055">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:355px;">MATERIALS</td>
			<td style="width:127px;">COMPANY</td>
			<td style="width:141px;">CATALOG NUMBER</td>
			<td style="width:432px;">COMMENTS</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMEM</td>
			<td>Corning</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Stem cell qualified FBS</td>
			<td>Gemini</td>
			<td>100-125</td>
			<td>Heat inactivated</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Penicillin/Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">L-alanyl L-glutamine</td>
			<td>Corning</td>
			<td>25-015-CI</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HEPES buffer&#160;</td>
			<td>Millipore</td>
			<td>TMS-003-C</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Non-Essential Amino Acids</td>
			<td>ThermoScientific</td>
			<td>SH30853.01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Gentamicin Regent Solution (50 mg/mL)</td>
			<td>Life Technologies&#160;</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#946;-mercaptoethanol (55 mM) in DPBS</td>
			<td>Life Technologies&#160;</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Filter Unit</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td>0.22&#956;m PES membrane</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cell Culture Grade Water</td>
			<td>Corning</td>
			<td>25-055-CM</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#945;-MEM&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>12000-022</td>
			<td>Powder, reconstitute per manufacture recommendation</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sodium bicarbonate&#160;</td>
			<td>Sigma</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">FBS</td>
			<td>ThermoScientific</td>
			<td>SH 30396.03</td>
			<td>Testing of individual lots required&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dimethyl Sulphoxide&#160;</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Recombinant Human Flt-3 Ligand</td>
			<td>R&#38;D Systems</td>
			<td>308-FK</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Recombinant Murine IL-7</td>
			<td>PeproTech</td>
			<td>217-17</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LIF&#160;</td>
			<td>Millipore</td>
			<td>ESG1107</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Utrapure water with 0.1% gelatin</td>
			<td>Millipore</td>
			<td>ES-006-B</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MEFs mitomycin C treated</td>
			<td>Millipore</td>
			<td>PMEF-CF</td>
			<td>Any mitotically arresteded MEFs can be used</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DPBS</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Trypsin EDTA, 1X</td>
			<td>Corning</td>
			<td>25-053-Cl</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cell strainer (40 &#956;m)</td>
			<td>Fisher</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tissue culture dish 100 X 20 mm</td>
			<td>BD Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Multiwell 6-well</td>
			<td>BD Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1.5 ml microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">15 ml centrifuge tubes</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">50 ml centrifuge tubes</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">5 ml Polystyrene Round-Bottom Tube with Cell-Strainer Cap</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td>Tubes for FACS&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">ES R1 cells</td>
			<td>ATCC</td>
			<td>SCRC-1011</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">OP9 cells</td>
			<td></td>
			<td></td>
			<td>Cells can be obtained from the Riken Laboratory Cell Repository (Japan).</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">OP9-DL1 cells</td>
			<td>&#160;&#160;&#160;&#160;</td>
			<td></td>
			<td>Cells can be requested from the Z&#250;&#241;iga-Pfl&#252;cker laboratory.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">FlowJo software&#160;</td>
			<td>Tree Star</td>
			<td></td>
			<td>FACS data analyses</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Flow Cytometer</td>
			<td>BD</td>
			<td></td>
			<td>FACScan, FACSCalibur and FACSVantage have been used in our lab</td>
		</tr>
	</tbody>
</table>

derivation of t cells in vitro from mouse embryonic stem cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

כאשר גדל על MEFs בנוכחות LIF, יכול להישמר mESCs במדינה שלא עברה התמיינות. בתנאים אידיאליים, הם מופיעים כמושבות קומפקטיות של תאים מוקפים בהילה נוצצת במיקרוסקופ לעומת שלב (איור 1). תרבויות אלה חייבים להיות במעקב יומי. בהתאם לנקודת המפגש של התאים, ניתן לשנות תקשורת או ניתן לפצל תאים. מושבות המסקלין שכנים לא צריכה להגיע לנקודת מגע זו עם זו. תרבות נורמלית, בריאה של mESCs מובחן היא נקודת התחלה חיונית במבחנת התמיינות תאי T. על ייזום שיתוף התרבות, מומלץ שהתאים להיות מחוברות באופן מלא מבלי להיות צפוף. זה כל כך את רוב התאים שנקטפו לזריעת תאים על גבי monolayers OP9 הם המסקלין (ולא תאים מזינים). אם הבדלים גדולים במפגש הם נצפו בין השיבוטים המסקלין השונים לבידול ביום 0, אז זה יהיה הטוב ביותר להסיר MEFs ידי מראש דבקות tisמנות תרבות לתבוע (כמו בשלב 3.4.3-3.4.6 באמצעות תקשורת בתאי גזע עובריים מלאה) לפני ספירת mESCs לזריעת שיתוף התרבות. תאי OP9 יש לשמור היטב לפני תחילת שיתוף התרבות (כפי שתואר בסעיף 2.4). בנוסף, הוא חשב שזה שOP9 תאים מאבדים עם תרבות ממושכת ו / או עליות בולטות בקצב ההתפשטות שלהם חלק מהנכסים שלהם 7 הימנעות יחסי פיצול מעבר 1:. 5 במהלך התחזוקה של OP9 תאים, ושלכת תרבויות OP9 רציפות לאחר שישה שבועות, יעזור להימנע ממלכודות אלה. בצעדי זריעת תאים והעברת תא ראשוניים של התרבות המשותפת (היום 0, 5, 8, 12, 16), monolayers OP9 צריך להיות בתוך טווח אופטימלי של מפגש. איור 2 מראה (איור 2 א) הנמוך וגבוה (איור 2B) מסתיים בטווח של מפגש תא OP9 מומלץ לשימוש כmonolayers שיתוף התרבות. דוגמא של צלחת מעל ומחוברות מוצגת באיור2C יור. כישלון לשמור על מפגש אופטימלי עשוי לגרום להיווצרות של תאי שומן (איור 2 ד) כי, בכמויות גדולות, עלול להשפיע לרעה על התרבות המשותפת. ביום 0, התרבות המשותפת הוא התחילה בOP9 תאים ולא OP9-DL1 תאים מאז היווצרות המזודרם היא מועדף על OP9 תאים. תאים מצופה בOP9-DL1 monolayers תאים המתחיל ביום 8 כדי לגרום לייצור תאי T. בעוד כמה שיבוטים המסקלין יהיו חזותי להציג היווצרות חזקה וכמותי (~ 90%) של מושבות כמו המזודרם-ליום 5 של תרבות המשותפת (איור 3A-B), אחרים יכולים להציג עיכובים בשלב זה (איור 3 ג-F). מתחת למיקרוסקופ, המושבות כמו המזודרם שמקורם בassay זה תוארו variably כדומה גלגלי עגלה או מכתשים (איור 4 א) או, מטר כוכבים או פרחים (איור 4). בעוד פרוטוקול OP9-DL1 פורסם משקף נורמה של פורם המזודרם כמותייםtion על ידי יום 5 של התרבות המשותפת, 7 אנו מוצאים כי לא כל השיבוטים ESC להשיג נורמה שבתוך מסגרת זמן זו. במקרים אלה, אנו משנים בינוניים ביום 5 ולחכות 1-2 ימים נוספים לפני ביצוע &quot;יום 5&quot; קציר התא / פרוטוקול העברה. המטרה של עיכוב זה היא לאפשר ~ 80-90% ממושבות בתאי גזע עובריים לmesodermal הפך חזותי לפני ההעברה. חשוב להבהיר כי דמות 80-90% זה משקפת להבחין קריטריון חזותי בקפדנות על ידי בדיקה פשוטה בניגוד השלב מיקרוסקופית של מורפולוגיה מושבה במנות התרבות המשותפת. זה מתייחס רק לחלקן של מושבות ESC דומות לאלו שמוצגים באיור 4. יתכן שחלק משיבוטי ESC לא יגיעו קריטריון חזותי זו, גם לאחר עיכוב של יומיים. במקרה כזה, ניתן להעשיר למבשרי mesodermal דם להרכיב באמצעות cytometry זרימת תא המיון לflk-1 + תאים, כפי שתוארו קודם לכן. 6,11 בכל מקרה, לsake נוחות מינוח, אנחנו &quot;לאפס את השעון&quot; ומתייחסים ליום העברת מושבה כמו המזודרם-כמו &quot;יום .5&quot; כאשר השיבוטים ESC מרובים שמתבצע מובחנים במקביל, זה אפשרי, כי שיתוף תרבויות מסוימות תהיה מוכנות למעבר ביום 5 בזמן, בעוד שאחרים משתרכים מאחור. במקרה זה, מומלץ לדחות את המעבר של כל שיתוף התרבויות עד השיבוטים איטיים יותר להדביק אחרים. נראה העיכוב לא לעשות שום נזק ל&quot; בזמן &quot;שיתוף התרבויות, אבל יתרונות הבידול הבא של השיבוטים איטיים הרבה. יום 8 (כלומר, שלושה ימים לאחר העברת המושבה mesodermal) רואה את ההופעה והצטברות של HPCs. HPCs נראה כמו אשכולות מבריקים של תאים שהם דבקו התאים מזינים OP9 (איור 5) באופן רופף. הליך כביסה זהיר, באמצעות פיפטה והתקשורת על הצלחת, תנתק ולאסוף HPCs תוך השארה(6A-C איור) monolayer OP9 בעיקר ללא פגע. זה אולי צעד הטכניקה רגישה ביותר של הפרוטוקול. זה לוקח חלק באימון כדי למצוא את התנועות המתאימות ובלחץ פליטת תקשורת כדי להשיג את האיזון הרצוי של קציר המקסימאלי של HPCs, עם הפרעה מינימאלית של שכבת המזין. זה מקובל, אם חלק מmonolayer OP9 מרים את, שכן הרוב של כל monolayer התועה יהיה שנתפס ברשת ניילון 40 מיקרומטר לאחר סינון. איור 6 ד תערוכות monolayer לאחר pipetting המופרז שמוביל לשיבוש monolayer גדול יותר ממה שצריך. במהלך שלב זה כביסה, זה חשוב לבדוק תחת מיקרוסקופ או אם לא כל HPCs כבר נאסף, ולהתאים את לחץ הכביסה בהתאם. התקדמות של שיתוף התרבות יכולה להיות במעקב על ידי cytometry זרימה. במיוחד כדי לנתח את צאצאי hematopoietic לא erythroid של ESC, חשוב השער הראשון בCD45 + תאי חיים.מסכי צעד זה את תאי OP9 מהניתוחים, ואילו שימוש בDAPI או צבע הכתמה גרעינית אחר מאפשר ההרחקה של תאי nonviable. ביום 12, אשכולות רבים של תאים קטנים, עגולים, מבריקים צריכים להיות גלויים. אין צורך לספור את התאים בשלב זה. אבל יש לנו ציינתי כי לחיות, ספירות אירוע cytometry זרימה מגודרת הן בדרך כלל בטווח של 1-3 x 10 5 יום 12 שיתוף תרבות לכל טוב. Cytometry זרימת מנתח של תאים מגודרת חיים המובחנים על monolayer OP9 יניב erythroid (neg CD45, TER119 +) וmonocytic (CD45 +, CD11b היי) שושלות (איור 7 א). ניתוח של תאי הבחנה על OP9-DL1 monolayers צריך לחשוף סמנים אופייניים שלילי כפול מסוג CD4 / CD8 חיים, CD45 + (DN) -1 (CD44 +, נג CD25, נג CD4, CD8 נג) וDN2 (CD44 +, CD25 +, נג CD4, CD8 נג) תאי T שלב (איור 7 </stro<html> ng&gt;). ביום 16, יש עלייה משמעותית בכמות תאים קטנים, עגולים, מבריקים בתרבויות. על OP9-DL1s, התקדמות מוצרי התמיינות תאי T ל( neg CD44, CD25 +, נג CD4, CD8 נג) DN3 וDN4 (neg CD44, CD25 נג, נג CD4, CD8 נג) שלבים. חלק מתאי T DP (מסוג CD4 +, CD8 +) יכולים גם להתחיל מתעוררים ביום 16 (איור 7). HPCs זורעים על OP9 תאים להניב תאי B (CD19 +) ביום 16. ביום 20 של התרבות המשותפת OP9-DL1-המסקלין, יהיו כמויות גדולות של תאי DP T וחיובי יחידים (SP) תאי CD8 + T הנוכחיות ( איור 7). איור 8 מספק תרשים המסכם את השלבים העיקריים של התהליך המתואר לעיל, ונקודות זמן ניתוח הציעו במהלך התרבות משותפת. ghres.jpg &quot;/&gt; תמונת איור 1 שלב מיקרוסקופ לעומת של mESCs מובחן (מושבות קצוות חדים) גדל על גבי monolayer MEF (הגדלה 100X). <img alt="איור 2" fo:content-width="3in" src="/files/ftp_upload/52119/52119fig2highres.jpg" width="600" /> איור 2:. תמונות שלב מיקרוסקופ לעומת של confluency monolayer OP9 () הנמוך ביותר ו( B) הרמות הגבוהות ביותר confluency מומלצת למעבר / זריעה (הגדלה 40X) שיתוף תרבות לדוגמא (ג) (הגדלה 40X מעל מחוברות monolayer OP9. ). ((הגדלה 200X יתר מחוברות monolayer OP9 D)) עם היווצרות adipocyte (גדולה, שלפוחית ​​המכילה, תאים). <img alt="איור 3" src="/files/ftp_upload/52119/52119fig3highres.jpg" /> איור 3: microscop בניגוד שלב<html> תמונות y של הקמת מושבה כמו המזודרם-ב&quot; יום 5 &quot;של התרבות המשותפת. () צפיות ב40X ו (ב) 100X של צלחות התרבות המשותפת עם&gt; 90% בידול המושבה mesodermal. צלחות אלה הן מוכנות ליום 5 מעבר. דוגמאות (CF) של צלחות יום 5 תרבות המשותפת הדורשות דחיית יום 1-2 לפני ההעברה (C &amp; E, הגדלה 40X, D &amp; F, הגדלת 100x). <img alt="איור 4" src="/files/ftp_upload/52119/52119fig4highres.jpg" /> איור 4: תמונות מיקרוסקופ לעומת שלב של מגוון רחב של תצורות מושבה כמו המזודרם-ביום 5 של התרבות המשותפת () מורפולוגיה המושבה מכונה &quot;מכתשים&quot; או (ב) מורפולוגיה המושבה המכונות. &quot;גלגל עגלה.&quot; כ&quot; מספרים &quot;או&quot; פרחים &quot;(הגדלה 200X). page = &quot;תמיד&quot;&gt; <img alt="איור 5" fo:content-width="4in" src="/files/ftp_upload/52119/52119fig5highres.jpg" width="600" /> תמונת איור 5 שלב מיקרוסקופ לעומת יום 8 צלחת שיתוף תרבות של עם אשכולות של HPCs הקטן, עגול, מבריק על monolayer OP9 (הגדלה 40X). <img alt="איור 6" src="/files/ftp_upload/52119/52119fig6highres.jpg" /> איור 6:.. Monolayer OP9 לאחר איסוף HPCs ביום התרבות משותפת 8 (AC) הטווח המתאים של שיבוש monolayer OP9 לאחר כביסה זהירה למסוק HPCs (ד &#39;) דוגמא של monolayer OP9 ששובש מעל מהיום ליום הליך קציר 8 HPC. <img alt="איור 7" src="/files/ftp_upload/52119/52119fig7highres.jpg" /> איור 7: cytometry זרימת נציג (FACS) מנתח בנקודות זמן מסוימים במהלךבידול המסקלין במבחנה. (א) מכתים לerythroid (משמאל), monocytic (באמצע) ותא B (מימין) מוצרים של התרבות המשותפת המסקלין-OP9 ביום 12 או 16 כפי שצוין. (B) מכתים לפיתוח תאי T בנקודות זמן המצוינת של המסקלין-OP9-DL1 שיתוף התרבות. אנא ראה טקסט עבור תיאורים מפורטים של immunophenotypes. <img alt="איור 8" src="/files/ftp_upload/52119/52119fig8highres.jpg" /> תרשים איור 8 מהצעדים של הליך שיתוף תרבות המסקלין-OP9. () צעדי העברת תא המפתח של שמונה הימים הראשונים של התרבות המשותפת. המספר המשוער של תאי זרע על תאי OP9 בימים אפס וחמש מצוינים. (ב &#39;) צעד העברת יום 8 ומוצרי ההתמיינות הצפויות זוהו על ידי cytometry זרימה בנקודתי זמן מרכזי של התרבות המשותפת. אנא ראה טקסט עבור תיאורים מפורטים של immunophenotypes.

Watch this Scientific Journal Video about Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells at JoVE.com

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

גזירה של תאי T<em&gt; במבחנה</em&gt; מתאי עכבר גזע העובריים

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

derivation-of-t-cells-in-vitro-from-mouse-embryonic-stem-cells

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

10.9K Views.  City University of New York. Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Video: Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

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 ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

התמיינות מכוונת של המושרה בתאי גזע pluripotent כלפי לימפוציטים-T

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

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 ...

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

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

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

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

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

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

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

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

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

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

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 ...

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 ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

במבחנה בידול של האדם CD4 + FOXP3 + מושרה תקינה בתאי T (iTregs) מ נאיבית CD4 + T תאים באמצעות פרוטוקול TGF-β-המכיל

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

במבחנה In Vivo למזהמים סביבתיים T, B והתאים מיאלואידית פעילות מדכא ומענים ההורמונאלית מן המקבלים

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

במבחנה הבידול של העכבר גורם גרנולוציט-מקרופאג-המושבה-מגרה (GM-CSF)-הפקת תאי T Helper (THGM)

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

טיהור והתרחבות של עכבר Invariant טבעי רוצח תאי T עבור במבחנה ו in vivo מחקרים

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...