The authors wish to thank Jennifer Strange and Greg Bauman for their assistance with Flow Cytometry analysis and sorting. This work was supported by the NIH Grant Number 2P20 RR020171 from the NCRR and by University of Kentucky startup funds to F.M.; G.I.E. acknowledges the support of the Presidential Graduate Fellowship and the Kentucky Opportunity Fellowship.

1. Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) from Buffy Coat
<ol>
 <li>Acquire one unit of buffy coat from hospital or nearby blood center location. Our blood center provides us with about 40-60 mL of buffy coat per unit obtained from normal blood donors.</li>
 <li>Pour blood into an autoclaved 500 mL glass bottle containing sterile PBS to dilute buffy coat. The final volume of PBS + buffy coat should be 250 mL.</li>
 <li>Fill ten 50 mL conical tubes each with 20 mL Lymphoprep solution.</li>
 <li>Ge...

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	<li>Powrie, F., Correa-Oliveira, R., Mauze, S., Coffman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+interactions+between+CD45RBhigh+and+CD45RBlow+CD4++T+cells+are+important+for+the+balance+between+protective+and+pathogenic+cell-mediated+immunity.">Regulatory interactions between CD45RBhigh and CD45RBlow CD4+ T cells are important for the balance between protective and pathogenic cell-mediated immunity.</a> The Journal of Experimental Medicine. 179, 589-600 (1994).</li><li>Tarbell, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell-expanded,+islet-specific+CD4++CD25++CD62L++regulatory+T+cells+restore+normoglycemia+in+diabetic+NOD+mice.">Dendritic cell-expanded, islet-specific CD4+ CD25+ CD62L+ regulatory T cells restore normoglycemia in diabetic NOD mice.</a> The Journal of Experimental Medicine. 204, 191-201 (2007).</li><li>Morgan, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+treatment+of+collagen-induced+arthritis+by+adoptive+transfer+of+CD25++regulatory+T+cells.">Effective treatment of collagen-induced arthritis by adoptive transfer of CD25+ regulatory T cells.</a> Arthritis and Rheumatism. 52, 2212-2221 (2005).</li><li>Taylor, P. A., Lees, C. J., Blazar, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+infusion+of+ex+vivo+activated+and+expanded+CD4(+)CD25(+)+immune+regulatory+cells+inhibits+graft-versus-host+disease+lethality.">The infusion of ex vivo activated and expanded CD4(+)CD25(+) immune regulatory cells inhibits graft-versus-host disease lethality.</a> Blood. 99, 3493-3493 (2002).</li><li>Ziegler, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FOXP3:+of+mice+and+men.">FOXP3: of mice and men.</a> Annual Review of Immunology. 24, 209-226 (2006).</li><li>Riley, J. L., June, C. H., Blazar, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+T+regulatory+cell+therapy:+take+a+billion+or+so+and+call+me+in+the+morning.">Human T regulatory cell therapy: take a billion or so and call me in the morning.</a> Immunity. 30, 656-6565 (2009).</li><li>Morgan, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+FOXP3+mRNA+is+not+confined+to+CD4+CD25++T+regulatory+cells+in+humans.">Expression of FOXP3 mRNA is not confined to CD4+CD25+ T regulatory cells in humans.</a> Human Immunology. 66, 13-20 (2005).</li><li>Wang, J., Huizinga, T. W. J., Toew, R. E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+Novo+Generation+and+Enhanced+Suppression+of+Human+CD4+CD25++Regulatory+T+Cells+by+Retinoic+Acid.">De Novo Generation and Enhanced Suppression of Human CD4+CD25+ Regulatory T Cells by Retinoic Acid.</a> Journal of Immunology. 183, 4119-4126 (2009).</li><li>Hori, S., Nomura, T., Sakaguchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+regulatory+T+cell+development+by+the+transcription+factor+Foxp3.">Control of regulatory T cell development by the transcription factor Foxp3.</a> Science (New York, N.Y.). 299, 1057-1061 (2003).</li><li>McHugh, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4(+)CD25(+)+immunoregulatory+T+cells:+gene+expression+analysis+reveals+a+functional+role+for+the+glucocorticoid-induced+TNF+receptor.">CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor.</a> Immunity. 16, 311-323 (2002).</li><li>Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunologic+Self-Tolerance+Maintained+by+Cd25+Cd4+Regulatory+T+Cells+Constitutively+Expressing+Cytotoxic+T+Lymphocyte-Associated+Antigen+4.">Immunologic Self-Tolerance Maintained by Cd25+Cd4+Regulatory T Cells Constitutively Expressing Cytotoxic T Lymphocyte-Associated Antigen 4.</a> The Journal of Experimental Medicine. 192, 303-310 (2000).</li><li>Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD127+expression+inversely+correlates+with+FoxP3+and+suppressive+function+of+human+CD4++T+reg+cells.">CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells.</a> The Journal of Experimental Medicine. 203, 1701-1711 (2006).</li><li>Schraven, B., Kalinke, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD28+superagonists:+what+makes+the+difference+in+humans.">CD28 superagonists: what makes the difference in humans.</a> Immunity. 28, 591-595 (2008).</li><li>Reneer, M. 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While Treg transfer holds enormous therapeutic promise in combating autoimmunity graft rejection and other immune or inflammatory-mediated disorders, methods for their efficient generation and stable maintenance have not yet been developed. As only 1-5% of circulating human T cells are Tregs, their controlled expansion and differentiation overcomes this paucity as a major deterrent of implementation of Tregs into the clinic. On the other hand, as we learned from the TGN1412 trial13 , it is scientifically and e...

<table border="1">
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Lymphoprep</td>
 <td>Axis-Shield</td>
 <td>1114547</td>
 <td>Keep at room temperature</td>
 </tr>
 <tr>
 <td>Refrigerated Bench-Top Centrifuge</td>
 <td>Eppendorf</td>
 <td>5810R</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bright-Line Hemocytometer</td>
 <td>Hausser Scientific</td>
 <td>3110</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EasySep Human CD4+ T Cell Enrichment Kit</td>
 <td>Stem Cell Technologies</td>
 <td>19052</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EasySep Human Memory CD4+ T Cell Enrichment Kit </td>
 <td>Stem Cell Technologies</td>
 <td>19157</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EasySep Human Na&iuml;ve CD4+ T Cell Enrichment Kit</td>
 <td>Stem Cell Technologies</td>
 <td>19155</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>The Big Easy EasySep Magnet</td>
 <td>Stem Cell Technologies</td>
 <td>18001</td>
 <td>Silver</td>
 </tr>
 <tr>
 <td>5 mL Round Bottom Polystyrene Tubes</td>
 <td>BD Falcon</td>
 <td>352008</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 mL Polypropylene Centrifuge Tubes</td>
 <td>VWR</td>
 <td>89004-368</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>14 mL Polypropylene Round-Bottom Tubes</td>
 <td>BD Falcon</td>
 <td>352059</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 mL Polypropylene Centrifuge Tubes</td>
 <td>VWR</td>
 <td>89004-364</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Human anti-CD3 Antibody</td>
 <td>Bio X Cell</td>
 <td>BE0001-2</td>
 <td>Clone: OKT3</td>
 </tr>
 <tr>
 <td>24 Well Cell Polystyrene Culture Plate</td>
 <td>BD Falcon</td>
 <td>353047</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>96 Well Round Bottom Tissue Culture Plate</td>
 <td>Grenier Bio-One</td>
 <td>650180</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RPMI 1640 with Glutamax-I and HEPES Buffer</td>
 <td>Gibco</td>
 <td>72400</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum (FBS)</td>
 <td>Gibco</td>
 <td>16000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&beta;-Mercaptoethanol</td>
 <td>Sigma</td>
 <td>M7522</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Recombinant Human TGF-&beta;1</td>
 <td>eBioscience</td>
 <td>14-8348-62</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Recombinant Human IL-2</td>
 <td>eBioscience</td>
 <td>14-8029-63</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin (BSA)</td>
 <td>MP Biomedicals Inc.</td>
 <td>810531</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.5 M EDTA</td>
 <td>Amresco</td>
 <td>E177</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mouse Anti-Human CD25-PE</td>
 <td>Miltenyi Biotec</td>
 <td>130-091-024</td>
 <td>Clone: 4E3</td>
 </tr>
 <tr>
 <td>Mouse Anti-Human CD45RA-PE-Cy5</td>
 <td>eBioscience</td>
 <td>15-0458-42</td>
 <td>Clone: HI100</td>
 </tr>
 <tr>
 <td>Mouse Anti-Human CD127-APC</td>
 <td>Miltenyi Biotec</td>
 <td>130-094-890</td>
 <td>Clone: MB15-18C9</td>
 </tr>
 <tr>
 <td>DNase II</td>
 <td>MP Biomedicals Inc.</td>
 <td>190370</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MoFlo Flow Cytometer</td>
 <td>Beckman Coulter</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FlowJo Software</td>
 <td>Tree Star, Inc.</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell Quest Pro Software</td>
 <td>BD Biosciences</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Newborn Calf Serum </td>
 <td>Gibco</td>
 <td>16010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Gibco</td>
 <td>25030</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>AIM-V</td>
 <td>Gibco</td>
 <td>0870112</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>CellTrace CFSE Cell Proliferation Kit</td>
 <td>Invitrogen</td>
 <td>C34554</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Treg Suppression Inspector Beads</td>
 <td>Miltenyi Biotec</td>
 <td>130-092-909</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Gibco</td>
 <td>15140</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>40 &mu;M Nylon Cell Strainer</td>
 <td>BD Falcon</td>
 <td>352340</td>
 <td>&nbsp;</td>
 </tr>
</table>

generation of induced regulatory t cells from primary human na&iuml;ve and memory t cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Watch this Scientific Journal Video about Generation of Induced Regulatory T Cells from Primary Human NaÃ¯ve and Memory T Cells at JoVE.com

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

generation-induced-regulatory-t-cells-from-primary-human-naive-memory

Research

JoVE Journal

Immunology and Infection

23.9K vistas.  University of Kentucky. Se describe un método para la generación de regulación, la memoria y el ingenuo células T de un donante de sangre humana única. Polarizada Tregs puede ser entonces en comparación con otros subconjuntos en una variedad de aplicaciones genéticas y funcionales con homogeneidad genética, incluyendo un ensayo de supresión también detallan aquí.