Christian Welker is funded by a grant provided by the J&#252;rgen-Manchot-Stiftung.

Ethic Statement: All procedures were carried out according to the Declaration of Helsinki and were approved by the Clinical Ethics Committee at the University of T&#252;bingen (projects 38/2009B02 and 470/2013B02).
1. Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
<ol>
	<li>Take 50-100 ml from a healthy volunteer via venipuncture using a 50 ml syringe containing 1,000 IU heparin sulfate and dilute the blood 1:2 with PBS (pH = 7.2).</li>
	<li>Carefully layer 35 ml of the blood:PBS solution on 15 ml blood separating solution such as Biocoll (d = 1.077 g/ml) in a 50 ml conical tube. Centrifuge for 15 min at 800 x g at RT. 
	NOTE: Brake must not be used.</li>
	<li>Collect the cells of the lymphocytic layer with a pipette and wash the cells twice in at least 50 ml PBS (centrifuge for 12 min; at 400 x g). Remove the supernatant after centrifugation with a pipette.</li>
	<li>Lyse remaining erythrocytes by suspending and incubating the lymphocytic fraction with 1-5 ml of a hypotonic buffer solution for 2-4 min. 
	NOTE: Duration of exposure and volume of lysing buffer depend on the used lysing buffer and on the amount of red blood cells left in the lymphocyte fraction.</li>
	<li>Dilute the cells with PBS (1:10) and centrifuge at 250 x g for 12 min and subsequently resuspend and wash the cell pellet once in 10 ml PBS at 300 x g for 12 min.</li>
	<li>Resuspend the lymphocytes in PBS and count the cells in a Neubauer counting chamber using Trypan blue solution. 
	NOTE: Usually, &#62;1 x 108 lymphocytes are received from 100 ml freshly drawn peripheral blood.</li>
</ol>
2. Isolation of V&#948;1 T Cells
<ol>
	<li>Take &#60;1 x 106 isolated lymphocytes for an initial FACS determining the frequency of V&#948;1 CD4 T cells. 
	NOTE: Population size of this T-cell entity may differ greatly between individuals and according to their immunologic state. Typically, about 1% of T cells are V&#948;1+ and about 1-5% of V&#948;1+ cells are CD4+.
	<ol>
		<li>Dilute the cells with 1 ml FACS-buffer, which consists of PBS containing 2% FCS and 5 mM EDTA. Centrifuge at 660 x g for 2 min and decant the supernatant. The reflux volume of about 100 &#181;l FACS buffer is sufficient for the subsequent staining. Briefly vortex and incubate the cells with 5 &#181;l Fc-Blocking Reagent for 5 min at RT for increased specificity of the FACS staining.</li>
		<li>Add human monoclonal antibodies directly to the cells without removing the Fc-Blocking Reagent. Make sure to stain the cells with antibodies against V&#948;1 (1:50), CD3 (1:50), CD4 (1:200), CD8 (1:200) and TCR&#945;&#946; (1:25). Prepare the isotype control with the corresponding immunoglobulin isotypes. Incubate cells with antibodies for 20 min at 4 &#176;C in the dark.</li>
		<li>Wash the cells in 1 ml FACS buffer (centrifuge for 2 min; at 660 x g), decant the supernatant and proceed to FACS acquisition in the remaining buffer volume.</li>
	</ol>
	</li>
	<li>Pelletize the cells (that are not used in the FACS analysis) by centrifugation for 12 min; at 300 g; 8 &#176;C. Remove the supernatant and resuspend the cell pelletin 60 &#181;l MACS-buffer per 1 x 107 cells. Add 20 &#181;l Fc-Blocking Reagent per 1 x 107 cells and incubate for 5 min at 4&#176;C.</li>
	<li>Add 10 &#181;l FITC-conjugated anti-human V&#948;1 antibody per 1 x 107 cells directly to the cells. Incubate for 12 min at 4 &#176;C in the dark.</li>
	<li>Wash the cells using 14 ml MACS-buffer (centrifuge for 12 min; at 300 g; 8 &#176;C). Discard supernatant completely and resuspend the cells in 80 &#181;l MACS buffer per 1x107 cells.</li>
	<li>Take 1-2 x 105 cells and dilute them in 100 &#181;l FACS buffer for verification of the labeling by FACS analysis. No further additives are needed for this step. Make sure that there is a sufficient difference in the brightness for V&#948;1- and V&#948;1+ cells. If this is not the case, repeat steps 2.3) and 2.4).</li>
	<li>Add 20 &#181;l anti-FITC microbeads per 1 x 107 cells to the remaining cells, mix well and incubate for 15 min in the dark at 4 &#176;C. Thereafter, wash the cells using at least 10 ml MACS buffer (centrifuge for 12 min, at 300 x g; 8 &#176;C).</li>
	<li>During the centrifugation, equilibrate the pre-cooled magnetic column placed in a magnet with 500 &#181;l MACS buffer.</li>
	<li>Resuspend the cells in 500 &#181;l MACS-buffer (for cell numbers higher than 1 x 108, scale up this volume accordingly) and apply the cells carefully onto the column. Collect the flow-through containing the V&#948;1- cells.</li>
	<li>Wash the column three times with 500 &#181;l MACS-buffer and collect the flow-through again in the same tube. Note that the reservoir must be empty before applying buffer onto the column.</li>
	<li>Remove the column from the magnetic device and place it into a 15 ml conical tube. Quickly flush the column with 1 ml MACS buffer by firmly pushing the plunger into the column. Collect the eluate in a tube. 
	NOTE: In order to obtain a purity of &#62;98%, it is usually necessary to repeat steps 2.7-2.9) with a second column.</li>
	<li>Verify the purity of the cells by FACS analysis18 with the same antibody panel as before (see 2.1.2) and count the cells.</li>
</ol>
3. Isolation of V&#948;1CD4+ T Cells
<ol>
	<li>Resuspend 25 &#181;l of CD4 beads with a vortex for &#62;30 sec. Wash the beads (the minimum amount as depicted by the manufacturer) in 1 ml MACS buffer in a 1.5 ml reaction tube by placing the tube into a magnet for 1 min. Discard the supernatant carefully with a small-scale pipette (200 &#181;l) and resuspend the beads in the original volume of MACS-buffer.</li>
	<li>Pelletize V&#948;1+ cells (centrifuge for 12 min; at 300 x g) and aspirate the supernatant and resuspend all the V&#948;1+ cells in 500 &#181;l MACS-buffer and add them to the tube containing the beads. Mix vigorously and incubate for 20 min at 4 &#176;C with constant tilting (e.g., in a rotator).</li>
	<li>Place the tube containing the cells in a magnet for 2 min. Make sure that there are no remnants in the lid. 
	NOTE: CD4+ cells will have bound the magnetic beads and attach to the side of the tube facing the magnet.</li>
	<li>Carefully open the lid while keeping the tube inside the magnetic device and collect the supernatant which contains the CD4- V&#948;1+ cells using a small-scale pipette. Place CD4- V&#948;1+ cells into a separate tube and place this tube into the magnet again to avoid possibly remaining CD4 cells or beads from the population.
	<ol>
		<li>Wash the CD4- cells in 5 ml MACS-buffer (centrifuge for 12 min; at 300 x g) and resuspend them in a concentration of 1 x 105 cells per 100 &#181;l media. CD4- cells can be readily cultivated under the conditions given below (4.2).</li>
	</ol>
	</li>
	<li>Place the tube with the CD4+ cell targets outside of the magnetic field, resuspend the cells in 500 &#181;l MACS-buffer and put them back into the magnetic device. Repeat steps 3.3-3.5 twice to obtain a higher purity. Remove supernatant by pipetting</li>
	<li>Resuspend the CD4+ cells in 100 &#181;l culture media (RPMI 1640, 10 % FCS, 1% L-Glutamine, 1% Penicillin/Streptomycin) and add 10 &#181;l of a bead-detaching solution. Incubate at RT for 45 min with constant tilting (e.g., in a rotator).</li>
	<li>Place the cells in a magnet for 1 min. Carefully collect the supernatant containing bead-free V&#948;1+CD4+ cells using a small-scale pipette.</li>
	<li>Outside of the magnet, resuspend the beads with 100 &#181;l culture media and repeat steps 3.6 and 3.7 twice to obtain higher cell numbers of CD4+ cells.</li>
	<li>Pelletize the cells (centrifuge at 300 x g, for 12 min) and discard the supernatant completely by pipetting. Resuspend cells in fresh, pre-warmed media and count them. Examine the purity of the V&#948;1+CD4+ cells by FACS analysis depicted in steps 2.1.1-2.1.3.</li>
</ol>
4. Single-cell Cloning by Limited Dilution
<ol>
	<li>Isolate peripheral blood mononuclear cells (PBMCs) from an allogeneic donor as depicted in 1.1-1.6.</li>
	<li>Irradiate 2.5 x 107 allogeneic PBMCs with 80 Gy in 25 ml culture media using &#947;-radiation.</li>
	<li>Add IL-2 (200 U/ml), IL-7 (20 ng/ml) and PHA (2 &#181;g/ml) to the irradiated feeder cells and distribute them in 96-well U-form plates, 5 x 104 feeder cells in 50 &#181;l per well.</li>
	<li>Dilute the V&#948;1+ CD4+ cells to a concentration of 0.3 cells per 50 &#181;l. Pipette 50 &#181;l of the cell solution to each well harboring the irradiated cells and cytokines. 
	NOTE: Thus, the cytokines are diluted to the final concentration of 100 U/ml IL-2, 10 ng/ml IL-7 and 1 &#181;g/ml PHA. Incubate the 96-well plates in an incubator at 37 &#176;C, 5% CO2 humidified atmosphere.
	<ol>
		<li>Optionally, cultivate remaining purified V&#948;1+ CD4+ cells as bulk culture under the same culture conditions as the clones.</li>
	</ol>
	</li>
	<li>Supply the cells every 3-4 days with cytokines and fresh media. For this, exchange half of the medium in the wells with 50 &#181;l fresh medium by pipetting. Add 2-fold concentration of cytokines to every supplementation. Add 5x104 irradiated feeder cells per well every other round of feeding.</li>
	<li>Using a microscope, observe first clones become visible after 3-4 weeks, as they metabolize and thus change the color of the culture medium and form colonies. 
	NOTE: For further cultivation, resuspend the clones well and transfer them to separate 96-well plates. Analyze the cells via FACS as indicated in 2.1.1-2.1.3. Clones kept under these conditions may eventually change their TCR from V&#948;1 to &#945;&#946;TCR. The time point for this transdifferentiation varies from clone to clone.</li>
</ol>

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	<li>Bhandoola, A., von, B. H., Petrie, H. T., 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=Commitment+and+developmental+potential+of+extrathymic+and+intrathymic+T+cell+precursors:+plenty+to+choose+from.">Commitment and developmental potential of extrathymic and intrathymic T cell precursors: plenty to choose from.</a> Immunity. 26 (6), 678-689 (2007).</li><li>Von, B. H., Melchers, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Checkpoints+in+lymphocyte+development+and+autoimmune+disease.">Checkpoints in lymphocyte development and autoimmune disease.</a> Nat. Immunol. 11 (1), 14-20 (2010).</li><li>Prinz, 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=Visualization+of+the+earliest+steps+of+gammadelta+T+cell+development+in+the+adult+thymus.">Visualization of the earliest steps of gammadelta T cell development in the adult thymus.</a> Nat. Immunol. 7 (9), 995-1003 (2006).</li><li>Ferrero, 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=TCRgamma+silencing+during+alphabeta+T+cell+development+depends+upon+pre-TCR-induced+proliferation.">TCRgamma silencing during alphabeta T cell development depends upon pre-TCR-induced proliferation.</a> J. Immunol. 177 (9), 6038-6043 (2006).</li><li>Krangel, M. S., Carabana, J., Abbarategui, I., Schlimgen, R., Hawwari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enforcing+order+within+a+complex+locus:+current+perspectives+on+the+control+of+V(D)J+recombination+at+the+murine+T-cell+receptor+alpha/delta+locus.">Enforcing order within a complex locus: current perspectives on the control of V(D)J recombination at the murine T-cell receptor alpha/delta locus.</a> Immunol. Rev. 200, 224-232 (2004).</li><li>Hawwari, A., Krangel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+rearranged+variable+gene+segments+in+directing+secondary+T+cell+receptor+alpha+recombination.">Role for rearranged variable gene segments in directing secondary T cell receptor alpha recombination.</a> Proc. Natl. Acad. Sci. U.S.A. 104 (3), 903-907 (2007).</li><li>Hawwari, A., Krangel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+TCR+delta+and+alpha+repertoires+by+local+and+long-distance+control+of+variable+gene+segment+chromatin+structure.">Regulation of TCR delta and alpha repertoires by local and long-distance control of variable gene segment chromatin structure.</a> J. Exp. Med. 202 (4), 467-472 (2005).</li><li>Hawwari, A., Bock, C., Krangel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+T+cell+receptor+alpha+gene+assembly+by+a+complex+hierarchy+of+germline+Jalpha+promoters.">Regulation of T cell receptor alpha gene assembly by a complex hierarchy of germline Jalpha promoters.</a> Nat. Immunol. 6 (5), 481-489 (2005).</li><li>Hager, E., Hawwari, A., Matsuda, J. L., Krangel, M. S., Gapin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+constraints+at+the+level+of+TCRalpha+rearrangement+impact+Valpha14i+NKT+cell+development.">Multiple constraints at the level of TCRalpha rearrangement impact Valpha14i NKT cell development.</a> J. Immunol. 179 (4), 2228-2234 (2007).</li><li>Linton, P. J., Dorshkind, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+lymphocyte+development+and+function.">Age-related changes in lymphocyte development and function.</a> Nat. Immunol. 5 (2), 133-139 (2004).</li><li>Sprent, J., Tough, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+life-span+and+memory.">Lymphocyte life-span and memory.</a> Science. 265 (5177), 1395-1400 (1994).</li><li>Guy-Grand, D., 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=Extrathymic+T+cell+lymphopoiesis:+ontogeny+and+contribution+to+gut+intraepithelial+lymphocytes+in+athymic+and+euthymic+mice.">Extrathymic T cell lymphopoiesis: ontogeny and contribution to gut intraepithelial lymphocytes in athymic and euthymic mice.</a> J. Exp. Med. 197 (3), 333-341 (2003).</li><li>McClory, S., 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=Evidence+for+a+stepwise+program+of+extrathymic+T+cell+development+within+the+human+tonsil.">Evidence for a stepwise program of extrathymic T cell development within the human tonsil.</a> J. Clin. Invest. 122 (4), 1403-1415 (2012).</li><li>Jbakhsh-Jones, S., Jerabek, L., Weissman, I. L., Strober, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrathymic+maturation+of+alpha+beta+T+cells+from+hemopoietic+stem+cells.">Extrathymic maturation of alpha beta T cells from hemopoietic stem cells.</a> J. Immunol. 155 (7), 3338-3344 (1995).</li><li>Garcia-Ojeda, 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=Stepwise+development+of+committed+progenitors+in+the+bone+marrow+that+generate+functional+T+cells+in+the+absence+of+the+thymus.">Stepwise development of committed progenitors in the bone marrow that generate functional T cells in the absence of the thymus.</a> J. Immunol. 175 (7), 4363-4373 (2005).</li><li>Arcangeli, M. L., 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=Extrathymic+hemopoietic+progenitors+committed+to+T+cell+differentiation+in+the+adult+mouse.">Extrathymic hemopoietic progenitors committed to T cell differentiation in the adult mouse.</a> J. Immunol. 174 (4), 1980-1988 (2005).</li><li>Maillard, 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=Notch-dependent+T-lineage+commitment+occurs+at+extrathymic+sites+following+bone+marrow+transplantation.">Notch-dependent T-lineage commitment occurs at extrathymic sites following bone marrow transplantation.</a> Blood. 107 (9), 3511-3519 (2006).</li><li>Ziegler, 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=Human+Peripheral+CD4(+)+Vdelta1(+)+gammadeltaT+Cells+Can+Develop+into+alphabetaT+Cells.">Human Peripheral CD4(+) Vdelta1(+) gammadeltaT Cells Can Develop into alphabetaT Cells.</a> Front Immunol. 5, 645 (2014).</li><li>Mollet, M., Godoy-Silva, R., Berdugo, C., Chalmers, J. 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The authors have nothing to disclose.

To study the phenotype, biology and function of a scarce (T-) cell entity, namely V&#948;1+CD4+ T cells, we used two markers: V&#948;1 and CD4 for its positive magnetic cell isolation. V&#948;1 is an orphan receptor, whereas CD4 is expressed on T helper cells, at a lower level on monocytes and dendritic cells, and at a very low level on hematopoietic progenitor cells.
Techniques for the enrichment and selection of cells in high purity include Fluorescence activated cell s...

In vertebrates, adaptive immunity that is structured in the cellular and a humoral part of immunity plays a major role in the defense against pathogens. The recognition of a wide range of antigens is mediated by hyperpolymorphic T- and B cell receptors (TCR/BCR), which with regard to T cells are assumed to be produced mainly in the thymus1. Thereto, hematopoietic stem cells (HSCs), derived from bone marrow, seed the thymus and differentiate along well-defined stages finally giving rise to all T cell lineages. Thymus seeding progenitors are CD4- and CD8- and thus constitute the immature, double negative (DN) thymocyte fraction. Thymus-derived signals then induce their lineage commitment and the differentiation into either &#945;&#946; or &#947;&#948; T cells. The expression of functionally rearranged TCR-&#947; and TCR-&#948; chain genes in DN2/3 thymocytes leads to &#947; &#948;TCR complexes, which drive cellular proliferation and promote differentiation into &#947; &#948;T cells2,3. In contrast, the rearrangement of a functional TCR-&#946; chain, that can pair with preT&#945; to build a preTCR pT, induces the transcriptional silencing of the TCR-&#947; chain in DN3 thymocytes and their transition into CD4+CD8+ double-positive thymocytes4. At this stage, recombination of the TCR-&#945; chain occurs, deleting the TCR-&#948; locus that nestles within the TCR-&#945; locus, thus abrogating the production a &#947;&#948;TCR in these cells irrevocably5-9. Rearranged &#945;&#946;TCRs are subsequently selected for their ability to bind self-MHC weakly (positive selection), which may not exceed a certain threshold to avoid autoimmunity (negative selection). According to their capacity of binding MHC class I or II, the selected &#945;&#946;T cells develop into single-positive CD4+ or CD8+ T cells, which exit the thymus as na&#239;ve T cells.
However, involution of the thymus starts early in life leading to exponentially reduced output of na&#239;ve T cells that is almost extinguished post-adolescence10. Nevertheless, the size of the T cell pool remains constant throughout life, which can be explained only in part by post-thymic homeostatic proliferation of T cells and the proliferation of long-lived immunologic memory11. Consequently, extrathymic T cell development must occur. Recent research has gained substantial attraction that characterized &#945;&#946;T cell progenitors, which-at extrathymic sites-gave rise to functional &#945;&#946; T cells12-17. Yet, detailed knowledge about extrathymic &#945;&#946;T cell precursors that independent from a thymus differentiate into &#945;&#946;T cells is as fragmentary as the background that we have on the route they take thereby.
We recently identified the small T-cell entity of V&#948;1+ CD4+&#947;&#948;T cells as an extrathymic &#945;&#946;T cell prognitor18, which when isolated from peripheral blood of healthy human donors can transdifferentiate into &#945;&#946;T cells in a mild inflammatory environment. Interestingly and contrary to the homeostatic proliferation of post-thymic T cells, transdifferentiation of V&#948;1 CD4+ cells generates new T cell receptors, thus broadening the repertoire diversity, so that potentially new antigens can be recognized and may protection the host against newly acquired pathogens. This adds to the plasticity of T cells and adds a so far unappreciated new pathway for extrathymic T cell development.
The quantitative isolation from lymphocytic sources, the generation of single-cell clones and their efficient expansion are essential for the objective to identify those markers and molecules that trigger this &#945;&#946;T cell precursor`s extrathymic development.

<table border="0" cellpadding="0" cellspacing="0" width="789">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biocoll Solution</td>
			<td style="width:93px;">Biochrom</td>
			<td style="width:119px;">L 6113</td>
			<td style="width:361px;">lymphocyte separating solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lysing Buffer</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>555899</td>
			<td>lysis of erythrocytes&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate-buffered Saline</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td style="width:119px;">D8537&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MACS buffer</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td>supplement with BSA and pre-cool before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BSA</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td>not mandatorily from this supplier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human Vd1 FITC (clone:&#160; TS8.2)</td>
			<td style="width:93px;">Thermo Scientific</td>
			<td>TCR2730</td>
			<td>not mandatorily from this supplier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD3 PerCP (clone: SK7)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>345766</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human TCRab PE (clone: T10B9.1A-31)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>555548</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD4 VioBlue (clone: M-T466)&#160;</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-097-333</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD8 APC-H7 (clone: SK1)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>641400</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Anti-FITC MultiSort Kit</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-058-701</td>
			<td>yields better results than anti-FITC MicroBeads</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MS columns</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-042-201</td>
			<td>pre-cool before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MiniMACS Separator</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CD4 Positive Isolation Kit</td>
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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.

Figure 1 depicts the different stages and the outcome of the isolation of V&#948;1 T cells from peripheral blood. Figure 1A shows a typical distribution of V&#948;1+ cells in CD3+ lymphocytes, as well as the co-receptor expression of the V&#948;1+ population. In this donor, the frequency of V&#948;1+ cells (red) is 2.3% of total lymphocyte counts and the CD4 expression (green) of V&#948;1+ lymphocytes is 2.6%. Altogether, the target ...

Watch this Scientific Journal Video about Isolation and Ex Vivo Culture of VÎ´1+CD4+Î³Î´ T Cells, an Extrathymic Î±Î²T-cell Progenitor at JoVE.com

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#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.

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 ...

isolation-ex-vivo-culture-v1-cd4-t-cells-an-extrathymic-t-cell

Research

JoVE Journal

Immunology and Infection

9.2K ビュー.  Children's University Hospital Tübingen. Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral Vδ1+CD4+ T cells that is, as we showed recently, an extrathymic αβ T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

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

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

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

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

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

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

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 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.

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

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

Isolation of Double Negative &amp;#945;&amp;#946; T Cells from the Kidney

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

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

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

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

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

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.

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

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

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

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

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

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...