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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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. 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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><tbody><tr><td>Biocoll Solution</td><td>Biochrom</td><td>L 6113</td><td>lymphocyte separating solution</td></tr><tr><td>Lysing Buffer</td><td>BD BioSciences</td><td>555899</td><td>lysis of erythrocytes&amp;nbsp;</td></tr><tr><td>Phosphate-buffered Saline</td><td>Sigma Aldrich</td><td>D8537&amp;nbsp;</td><td></td></tr><tr><td>MACS buffer</td><td>Miltenyi Biotec</td><td>130-091-222</td><td>supplement with BSA and pre-cool before use</td></tr><tr><td>BSA</td><td>Miltenyi Biotec</td><td>130-091-376</td><td>not mandatorily from this supplier</td></tr><tr><td>anti-human Vd1 FITC (clone:&amp;nbsp; TS8.2)</td><td>Thermo Scientific</td><td>TCR2730</td><td>not mandatorily from this supplier</td></tr><tr><td>anti-human CD3 PerCP (clone: SK7)</td><td>BD BioSciences</td><td>345766</td><td>not mandatorily from this supplier or this flurochrome</td></tr><tr><td>anti-human TCRab PE (clone: T10B9.1A-31)</td><td>BD BioSciences</td><td>555548</td><td>not mandatorily from this supplier or this flurochrome</td></tr><tr><td>anti-human CD4 VioBlue (clone: M-T466)&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-097-333</td><td>not mandatorily from this supplier or this flurochrome</td></tr><tr><td>anti-human CD8 APC-H7 (clone: SK1)</td><td>BD BioSciences</td><td>641400</td><td>not mandatorily from this supplier or this flurochrome</td></tr><tr><td>Anti-FITC MultiSort Kit</td><td>Miltenyi Biotec</td><td>130-058-701</td><td>yields better results than anti-FITC MicroBeads</td></tr><tr><td>MS columns</td><td>Miltenyi Biotec</td><td>130-042-201</td><td>pre-cool before use</td></tr><tr><td>MiniMACS Separator</td><td>Miltenyi Biotec</td><td>130-042-102</td><td></td></tr><tr><td>CD4 Positive Isolation Kit</td><td>life technologies</td><td>11331D</td><td></td></tr></tbody></table>

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 ...

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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

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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

9.2K Views.  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.

Video: Isolation and Ex Vivo Culture of Vδ1+CD4+γδ T Cells, an Extrathymic αβT-cell Progenitor

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Developmental Biology

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (&#947;&#948;) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure &#947;&#948; T cells drug product for first-in-human use of allogeneic &#947;&#948; T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. 
The lymphocyte-rich fraction is utilized, and the &#947;&#948; T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of &#947;&#948; T cells, a clinical-grade magnetic cell-separation device and TCR&#945;&#946; beads are used to deplete contaminating T-cell receptor (TCR)&#945;&#946; T cells. The highly enriched &#947;&#948; T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched &#947;&#948; T cells in co-culture with the aAPCs facilitates the manufacture of highly pure &#947;&#948; T cells with an average fold expansion of &gt;229,000-fold from healthy donor blood.

Expansion and Enrichment of Gamma-Delta &#947;&#948; T Cells from Apheresed Human Product

Presented is a protocol for the expansion of Gamma-Delta (&#947;&#948;) T cell drug product. Lymphocytes are isolated by elutriation and &#947;&#948;-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The &#947;&#948; cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...

Cancer Research

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have been conducive to downstream in vivo functional analysis of TCR heterodimers. We have developed an improved protocol based on a two-step multiplex-nested PCR, which results in a PCR product that spans entire variable regions of a human TCR alpha and beta chains. By identifying unique restriction sites and incorporating them into the PCR primers, we have made the PCR product compatible with direct sub-cloning into the template retroviral vector. The resulting retroviral construct encodes a chimeric human/mouse TCR with a mouse intracellular domain, which is functional in mouse cells or in in vivo mouse models. Overall, the protocol described here combines human single cell paired TCR alpha and beta chain identification with streamlined generation of retroviral vectors readily adaptable for in vitro and in vivo TCR expression. The video and the accompanying material are designed to give a highly detailed description of the single cell PCR, so that the critical steps can be followed and potential pitfalls avoided. Additionally, we provide a detailed description of the cloning steps necessary to generate the expression vector. Once mastered, the whole procedure from single cell sorting to TCR expression could be performed in a short two-week period.

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

The current protocol combines single cell paired human TCR alpha and beta chain sequencing with streamlined generation of retroviral vectors compatible with in vitro and in vivo TCR expression.

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have ...

Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high mortality, despite new therapeutic options such as transcatheter aortic valve replacement (TAVR). 
The complete pathways that start with valve calcification and lead to severe aortic stenosis remain only partly understood. By providing a close representation of the aortic valve cells in vivo, the assaying of T lymphocytes from stenotic valve tissue could be an efficient way to clarify their role in the development of calcification. After surgical excision, the fresh aortic valve sample is dissected in small pieces and the T lymphocytes are cultured, cloned then analyzed using fluorescence activated cell sorting (FACS).
The staining procedure is simple and the stained tubes can also be fixed using 0.5% of paraformaldehyde and analyzed up to 15 days later. The results generated from the staining panel can be used to track changes in T cell concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific T cell subtypes of interest. In this study, we show the isolation of T cells, performed on fresh calcified aortic valve samples and the steps of analyzing T cell clones using flow cytometry to further understand the role of adaptive immunity in CAVD pathophysiology.

In this study, we describe the process of T lymphocyte isolation from fresh samples of calcified aortic valves and the analytical steps of T cell-cloning for the characterization of the adaptive leukocyte subsets by using flow cytometry analysis.

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high ...

High-Resolution Imaging Technique for Detecting Ca2+ Microdomains in T Cells

Imaging Initial Ca2+ Microdomains in Primary T Cells

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and might already determine the fate of a T cell. Upon T cell receptor activation, NAADP is formed rapidly, binding to NAADP binding proteins (HN1L/JPT2, LSM12) and their respective receptors (RyR1, TPC2) sitting on intracellular Ca2+ stores, like the ER and lysosomes, and leading to subsequent release and elevation of [Ca2+]i. To capture these fast and dynamically occurring Ca2+ signals, we developed a high-resolution imaging technique using a combination of two Ca2+ indicators, Fluo-4 AM and FuraRed AM. For postprocessing, an open-source, semi-automated Ca2+ microdomain detection approach was developed based on the programming language Python. Using this workflow, we are able to reliably detect Ca2+ microdomains on a subcellular level in primary murine and human T cells in high temporal and spatial resolution fluorescence videos. This method can also be applied to other cell types, like NK cells and murine neuronal cell lines.

Author Spotlight: Unraveling the Initial Activation of the Adaptive Immune Response for Therapeutic Intervention

Here, we provide a comprehensive protocol to resolve initial local Ca2+ signals, known as Ca2+ microdomains, in primary murine and human T cells using fluorescence microscopy. This protocol serves as a valuable resource for researchers examining Ca2+ signaling pathways within immune cells and to further unravel their function.

Local, sub-second Ca2+ signals, termed Ca2+ microdomains, are highly dynamic and short-lived Ca2+ signals, which result in a global [Ca2+]i elevation and ...

An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells

Source: Ghassemi, S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59949">Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy.</a> J. Vis. Exp. (2019)
This video demonstrates a technique to manufacture chimeric antigen receptor (CAR) T cells ex vivo. Human T cells are incubated with magnetic beads coated with anti-CD3 and anti-CD28 antibodies, along with a medium supplemented with interleukin-2 (IL-2), leading to T cell activation and proliferation. Transgenic lentiviral vectors are introduced, allowing the transgenic RNA to integrate into the host genome and express the transgene. This results in the transduced T cells expressing chimeric antigen receptors (CARs) on their surface. The T cells are harvested at defined time points to assess CAR expression and cryopreservation.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. T Cell Activation, Transduction, ...

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An In Vitro Technique to Differentiate GM-CSF Producing T Helper Cells from Na&iuml;ve T Cells

Source: Lu, Y., et al.&#160;<a href="https://appjove.unicartagenaproxy.elogim.com/v/58087">In Vitro&#160;Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells.</a>&#160;J. Vis. Exp. (2018).
This video demonstrates the in vitro differentiation of granulocyte-macrophage-colony-stimulating factor or GM-CSF-producing T helper cells (THGM). Isolated na&#239;ve T cells are stimulated for differentiation into THGM, which is confirmed by detecting high intracellular GM-CSF production and a low expression of other T helper cell-specific cytokines.

Source: Lu, Y., et al.&#160;In Vitro&#160;Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) ...

Isolation and Ex Vivo Culture of Vδ1⁺CD4⁺γδ T Cells, an Extrathymic αβT-cell Progenitor

Summary

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Chapters in this video

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Isolation and Th17 Differentiation of Naïve CD4 T Lymphocytes

Expansion and Enrichment of Gamma-Delta (γδ) T Cells from Apheresed Human Product

Mouse Naïve CD4⁺ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat

Imaging Initial Ca²⁺ Microdomains in Primary T Cells

An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells

An In Vitro Technique to Differentiate GM-CSF Producing T Helper Cells from Naïve T Cells