Nina Nagel is gratefully acknowledged for technical assistance during the video shoot and experimental preparation. We thank Eva-Maria Weiss for help with the intracellular FOXP3 staining protocol and Elisabeth Suri-Payer and Nina Oberle for establishing the nTreg isolation protocol. Matilda Eriksson and Peri Noori are acknowledged for laboratory management. 
Funding: A.S. was supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme, the Dr. &#197;ke Olsson Foundation and KI research foundations; A.S. and J.T. were supported by a CERIC (Center of Excellence for Research on Inflammation and Cardiovascular disease) grant, J.T. was supported by Vetenskapsr&#229;det Medicine and Health (Dnr 2011-3264), Torsten S&#246;derberg Foundation, FP7 STATegra, AFA Insurance and Stockholm County Council.

<ol>
	<li>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=Regulatory+T+cells:+history+and+perspective.">Regulatory T cells: history and perspective.</a> Methods Mol.Biol. 707, 3-17 (2011).</li><li>DeLeeuw, R. J., Kost, S. E., Kakal, J. A., Nelson, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prognostic+value+of+FoxP3++tumor-infiltrating+lymphocytes+in+cancer:+A+critical+review+of+the+literature.">The prognostic value of FoxP3+ tumor-infiltrating lymphocytes in cancer: A critical review of the literature.</a> Clin Can Res. 18 (11), 3022-3029 (2012).</li><li>Liu, C., Workman, C. J., Vignali, D. A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Regulatory+T+Cells+in+Tumors.">Targeting Regulatory T Cells in Tumors.</a> FEBS J. , (2016).</li><li>Trzonkowski, P., 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=First-in-man+clinical+results+of+the+treatment+of+patients+with+graft+versus+host+disease+with+human+ex+vivo+expanded+CD4+CD25+C.">First-in-man clinical results of the treatment of patients with graft versus host disease with human ex vivo expanded CD4+CD25+C.</a> Clin. Immunol. 133, 22-26 (2009).</li><li>Brunstein, C. G., 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=Infusion+of+ex+vivo+expanded+T+regulatory+cells+in+adults+transplanted+with+umbilical+cord+blood:+safety+profile+and+detection+kinetics.">Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics.</a> Blood. 117, 1061-1070 (2011).</li><li>Di Ianni, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tregs+prevent+GVHD+and+promote+immune+reconstitution+in+HLA-haploidentical+transplantation.">Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation.</a> Blood. 117, 3921-3928 (2011).</li><li>Edinger, M., Hoffmann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+T+cells+in+stem+cell+transplantation:+strategies+and+first+clinical+experiences.">Regulatory T cells in stem cell transplantation: strategies and first clinical experiences.</a> Curr. Opin. Immunol. 23, 679-684 (2011).</li><li>Bluestone, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+1+diabetes+immunotherapy+using+polyclonal+regulatory+T+cells.">Type 1 diabetes immunotherapy using polyclonal regulatory T cells.</a> Science Transl Med. 7 (315), 189 (2015).</li><li>Haribhai, 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=A+central+role+for+induced+regulatory+T+cells+in+tolerance+induction+in+experimental+colitis.">A central role for induced regulatory T cells in tolerance induction in experimental colitis.</a> J. Immunol. 182, 3461-3468 (2009).</li><li>Haribhai, 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=A+requisite+role+for+induced+regulatory+T+cells+in+tolerance+based+on+expanding+antigen+receptor+diversity.">A requisite role for induced regulatory T cells in tolerance based on expanding antigen receptor diversity.</a> Immunity. 35, 109-122 (2011).</li><li>Abbas, A. K., 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=Regulatory+T+cells:+recommendations+to+simplify+the+nomenclature.">Regulatory T cells: recommendations to simplify the nomenclature.</a> Nat.Immunol. 14, 307-308 (2013).</li><li>Curotto de Lafaille, M. A., Lafaille, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+and+adaptive+foxp3++regulatory+T+cells:+more+of+the+same+or+a+division+of+labor.">Natural and adaptive foxp3+ regulatory T cells: more of the same or a division of labor.</a> Immunity. 30, 626-635 (2009).</li><li>Huehn, J., Polansky, J. K., Hamann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+control+of+FOXP3+expression:+the+key+to+a+stable+regulatory+T-cell+lineage.">Epigenetic control of FOXP3 expression: the key to a stable regulatory T-cell lineage.</a> Nat. Rev. Immunol. 9, 83-89 (2009).</li><li>Schmitt, E. G., Williams, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+function+of+induced+regulatory+T+cells.">Generation and function of induced regulatory T cells.</a> Front Immunol. 4, 152 (2013).</li><li>Pillai, V., Ortega, S. B., Wang, C. K., Karandikar, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+regulatory+T-cells:+a+state+attained+by+all+activated+human+T-cells.">Transient regulatory T-cells: a state attained by all activated human T-cells.</a> Clin.Immunol. 123, 18-29 (2007).</li><li>Wang, J., Ioan-Facsinay, A., vander Voort, E. I. H., Huizinga, T. W., Toes, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+expression+of+FOXP3+in+human+activated+nonregulatory+CD4++T+cells.">Transient expression of FOXP3 in human activated nonregulatory CD4+ T cells.</a> Eur. J. Immunol. 37, 129-138 (2007).</li><li>Josefowicz, S. Z., Rudensky, A. <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+lineage+commitment+and+maintenance.">Control of regulatory T cell lineage commitment and maintenance.</a> Immunity. 30, 616-625 (2009).</li><li>Sun, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+intestine+lamina+propria+dendritic+cells+promote+de+novo+generation+of+Foxp3+T+reg+cells+via+retinoic+acid.">Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid.</a> J.Exp.Med. 204, 1775-1785 (2007).</li><li>Coombes, J. 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=A+functionally+specialized+population+of+mucosal+CD103++DCs+induces+Foxp3++regulatory+T+cells+via+a+TGF-beta+and+retinoic+acid-dependent+mechanism.">A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-beta and retinoic acid-dependent mechanism.</a> J. Exp. Med. 204, 1757-1764 (2007).</li><li>Kang, S. G., Lim, H. W., Andrisani, O. M., Broxmeyer, H. E., Kim, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+A+metabolites+induce+gut-homing+FoxP3++regulatory+T+cells.">Vitamin A metabolites induce gut-homing FoxP3+ regulatory T cells.</a> J. Immunol. 179, 3724-3733 (2007).</li><li>Mucida, 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=Retinoic+acid+can+directly+promote+TGF-beta-mediated+Foxp3(+)+Treg+cell+conversion+of+naive+T+cells.">Retinoic acid can directly promote TGF-beta-mediated Foxp3(+) Treg cell conversion of naive T cells.</a> Immunity. 30, 471-472 (2009).</li><li>Schmidt, A., Eriksson, M., Shang, M. -. M., Weyd, H., Tegn&#233;r, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Analysis+of+Protocols+to+Induce+Human+CD4+Foxp3++Regulatory+T+Cells+by+Combinations+of+IL-2,+TGF-beta,+Retinoic+Acid,+Rapamycin+and+Butyrate.">Comparative Analysis of Protocols to Induce Human CD4+Foxp3+ Regulatory T Cells by Combinations of IL-2, TGF-beta, Retinoic Acid, Rapamycin and Butyrate.</a> PloS one. 11 (2), 0148474 (2016).</li><li>Furusawa, Y., Obata, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=regulatory+T+cells.">regulatory T cells.</a> Nature. 504, 446-450 (2013).</li><li>Arpaia, N., 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=Metabolites+produced+by+commensal+bacteria+promote+peripheral+regulatory+T-cell+generation.">Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation.</a> Nature. 504, 451-455 (2013).</li><li>Battaglia, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapamycin+promotes+expansion+of+functional+CD4+CD25+FOXP3++regulatory+T+cells+of+both+healthy+subjects+and+type+1+diabetic+patients.">Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients.</a> J. Immunol. 177, 8338-8347 (2006).</li><li>Hippen, K. 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=Massive+ex+vivo+expansion+of+human+natural+regulatory+T+cells+(T(regs))+with+minimal+loss+of+in+vivo+functional+activity.">Massive ex vivo expansion of human natural regulatory T cells (T(regs)) with minimal loss of in vivo functional activity.</a> Sci. Transl. Med. 3, 41 (2011).</li><li>Schmidt, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+macrophages+induce+CD4(+)Foxp3(+)+regulatory+T+cells+via+binding+and+re-release+of+TGF-&#946;.">Human macrophages induce CD4(+)Foxp3(+) regulatory T cells via binding and re-release of TGF-&#946;.</a> Immunol cell biol. , (2016).</li><li>Baron, U., 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=DNA+demethylation+in+the+human+FOXP3+locus+discriminates+regulatory+T+cells+from+activated+FOXP3(+)+conventional+T+cells.">DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3(+) conventional T cells.</a> Eur. J. Immunol. 37, 2378-2389 (2007).</li><li>Schmidt, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+regulatory+T+cells+rapidly+suppress+T+cell+receptor-induced+Ca(2+),+NF-kappaB,+and+NFAT+signaling+in+conventional+T+cells.">Human regulatory T cells rapidly suppress T cell receptor-induced Ca(2+), NF-kappaB, and NFAT signaling in conventional T cells.</a> Sci. Signal. 4, 90 (2011).</li><li>Ohkura, N., 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=T+cell+receptor+stimulation-induced+epigenetic+changes+and+Foxp3+expression+are+independent+and+complementary+events+required+for+Treg+cell+development.">T cell receptor stimulation-induced epigenetic changes and Foxp3 expression are independent and complementary events required for Treg cell development.</a> Immunity. 37, 785-799 (2012).</li><li>Hill, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+enhances+Foxp3+induction+indirectly+by+relieving+inhibition+from+CD4+CD44hi+Cells.">Retinoic acid enhances Foxp3 induction indirectly by relieving inhibition from CD4+CD44hi Cells.</a> Immunity. 29, 758-770 (2008).</li><li>Yang, R., 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=Hydrogen+Sulfide+Promotes+Tet1-+and+Tet2-Mediated+Foxp3+Demethylation+to+Drive+Regulatory+T+Cell+Differentiation+and+Maintain+Immune+Homeostasis.">Hydrogen Sulfide Promotes Tet1- and Tet2-Mediated Foxp3 Demethylation to Drive Regulatory T Cell Differentiation and Maintain Immune Homeostasis.</a> Immunity. 43 (2), 251-263 (2015).</li><li>Yue, X., Trifari, 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=Control+of+Foxp3+stability+through+modulation+of+TET+activity.">Control of Foxp3 stability through modulation of TET activity.</a> The Journal of experimental medicine. 213 (3), 377-397 (2016).</li><li>Sasidharan Nair, V., Song, M. H., Oh, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+C+Facilitates+Demethylation+of+the+Foxp3+Enhancer+in+a+Tet-Dependent+Manner.">Vitamin C Facilitates Demethylation of the Foxp3 Enhancer in a Tet-Dependent Manner.</a> J Immunol. 196 (5), 2119-2131 (2016).</li><li>Geiger, T. L., Tauro, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+and+nurture+in+Foxp3+++regulatory+T+cell+development,+stability,+and+function.">Nature and nurture in Foxp3 + regulatory T cell development, stability, and function.</a> Hum Immunol. 73 (3), 232-239 (2012).</li><li>Gu, J., 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=TGF-&#946;-induced+CD4+Foxp3++T+cells+attenuate+acute+graft-versus-host+disease+by+suppressing+expansion+and+killing+of+effector+CD8++cells.">TGF-&#946;-induced CD4+Foxp3+ T cells attenuate acute graft-versus-host disease by suppressing expansion and killing of effector CD8+ cells.</a> J Immunol. 193 (7), 3388-3397 (2014).</li><li>Brusko, T. M., Hulme, M. A., Myhr, C. B., Haller, M. J., Atkinson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+In+Vitro+Suppressive+Capacity+of+Regulatory+T+Cells.">Assessing the In Vitro Suppressive Capacity of Regulatory T Cells.</a> Immunol Invest. 36 (5-6), 607-628 (2007).</li><li>McMurchy, A. N., Levings, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+assays+with+human+T+regulatory+cells:+a+technical+guide.">Suppression assays with human T regulatory cells: a technical guide.</a> Eur J immunol. 42 (1), 27-34 (2012).</li><li>Mutis, T., 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+regulatory+T+cells+control+xenogeneic+graft-versus-host+disease+induced+by+autologous+T+cells+in+RAG2-/-gammac-/-+immunodeficient+mice.">Human regulatory T cells control xenogeneic graft-versus-host disease induced by autologous T cells in RAG2-/-gammac-/- immunodeficient mice.</a> Clin cancer res. 12 (18), 5520-5525 (2006).</li><li>Tran, D. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+suppression+assay+for+functional+assessment+of+human+regulatory+T+cells.">In vitro suppression assay for functional assessment of human regulatory T cells.</a> Meth mol biol. 979, 199-212 (2013).</li><li>Oberle, N., Eberhardt, N., Falk, C. S., Krammer, P. H., Suri-Payer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Suppression+of+Cytokine+Transcription+in+Human+CD4+CD25+T+Cells+by+CD4+Foxp3++Regulatory+T+Cells:+Independence+of+IL-2+Consumption,+TGF-beta,+and+Various+Inhibitors+of+TCR+Signaling.">Rapid Suppression of Cytokine Transcription in Human CD4+CD25 T Cells by CD4+Foxp3+ Regulatory T Cells: Independence of IL-2 Consumption, TGF-beta, and Various Inhibitors of TCR Signaling.</a> J. Immunol. 179, 3578-3587 (2007).</li><li>Shevach, E. M., Thornton, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=tTregs,+pTregs,+and+iTregs:+similarities+and+differences.">tTregs, pTregs, and iTregs: similarities and differences.</a> Immunol. Rev. 259, 88-102 (2014).</li></ol>

The authors have nothing to disclose.

The described protocol enables the robust induction of human CD4+FOXP3+ iTregs from human na&#239;ve CD4+ T cells. It includes a new protocol that we described recently, using a combination of TGF-&#946;, ATRA and rapamycin, for induction of iTregs with superior in vitro suppressive function22. Compared to other published protocols, another advantage is the induction of different iTreg populations in parallel by different protocols, which enables the direct comparison of effects of certain ...

CD4+CD25+FOXP3+ regulatory T cells (Tregs) suppress other immune cells and are critical mediators of peripheral tolerance, preventing autoimmunity and excessive inflammation1. The importance of Tregs is exemplified by the human disease immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX), in which loss of Tregs due to mutations in the `master&#180; Treg transcription factor forkhead box P3 (FOXP3) leads to severe systemic autoimmune disease, lethal at an early age. However, Tregs act as a double-edged sword in the immune system as they can also hamper anti-tumor immunity in certain settings2. Therapeutic manipulation of Treg number and function is therefore subject to numerous clinical investigations. In cancer, depletion of Tregs can be desirable and some success of clinical approaches encourages further research3. In autoimmune and inflammatory diseases, in addition to therapeutic effects of Tregs in several mouse disease models, recent first in-man trials of adoptive Treg transfer to prevent graft-versus-host disease (GvHD)4-7 and to assess safety in treating type 1 diabetes8 showed very promising outcomes.
Naturally occurring Tregs (nTregs) comprise thymic-derived tTregs and peripherally induced pTregs, with non-redundant essential functions in maintaining health9-11. However, nTreg numbers are limited, encouraging the complementary approach of inducing Tregs (iTregs) in vitro from na&#239;ve T cell precursors12. Still stability of iTregs, presumably due to lack of demethylation in the so-called Treg-specific demethylated region (TSDR) in the FOXP3 gene locus13, remains a concern and several studies indicate that in vivo induced Tregs are more stable14.
To date, FOXP3 remains the best protein marker for Tregs but it is not absolutely specific because human conventional CD4+CD25- T cells transiently express intermediate levels of FOXP3 upon activation15,16. Although significant progress has been made in elucidating the regulation of FOXP3 expression, much remains to be discovered regarding the induction, stability and function of FOXP3 particularly in human cells. Despite differences to nTregs, in vitro induced FOXP3+ CD4+ T cells can be used as a model system to study molecular mechanisms of FOXP3 induction and as a starting point to develop protocols in the future that allow for generation of iTregs that are more similar to in vivo generated Tregs, which could be applicable for adoptive transfer strategies in the future.
There is no `gold standard&#180; protocol to induce human iTregs, and current protocols have been developed based on mimicking Treg-inducing conditions in vivo: interleukin 2 (IL-2) and transforming growth factor &#946; (TGF-&#946;) signaling are crucial for FOXP3 induction in vivo17, and all-trans retinoic acid (ATRA) &#8212; which is produced in vivo by gut-associated dendritic cells &#8212; is frequently used to enhance FOXP3 induction in vitro18-21. We have developed additional human Treg-inducing protocols using butyrate22, a gut microbiota-derived short-chain fatty acid that was recently shown to augment murine Treg induction23,24. We also recently established a new protocol for generation of iTregs with superior suppressive function in vitro by using a combination of TGF-&#946;, ATRA and rapamycin22, the latter being a clinically approved mammalian target of rapamycin (mTOR) inhibitor that is known to promote FOXP3 maintenance during human Treg expansion25,26.
This method describes the reproducible in vitro generation of human CD4+FOXP3+ iTregs using a set of different conditions, and their subsequent phenotyping by flow cytometry and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to reveal protocol-specific patterns of expression of FOXP3 and other Treg signature molecules such as CD25, CTLA-4, EOS, as well as repression of IFN-&#947; and SATB1 expression22. The generated cell populations can be used for functional assays regarding suppressive activity or for molecular studies, either concerning general FOXP3 regulators or to study effects specific to certain compounds such as butyrate or rapamycin. Further understanding of molecular mechanisms driving Treg differentiation is highly relevant for future therapeutic approaches in autoimmunity or cancer to specifically target molecules involved in Treg generation and function.

<table><tbody><tr><td>All-trans retinoic acid</td><td>Sigma Aldrich</td><td>R2625-50MG&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>anit-human Foxp3-APC clone 236A/E7</td><td>eBioscience</td><td>17-4777-42</td><td></td></tr><tr><td>anti-human CD25 microbeads</td><td>Miltenyi Biotec</td><td>130-092-983</td><td></td></tr><tr><td>anti-human CD25-PE</td><td>Miltenyi Biotec</td><td>130-091-024</td><td></td></tr><tr><td>anti-human CD28 antibody, LEAF Purified&amp;nbsp;</td><td>Biolegend</td><td>302914</td><td></td></tr><tr><td>anti-human CD3 Antibody, LEAF Purified&amp;nbsp;</td><td>Biolegend</td><td>317315</td><td></td></tr><tr><td>anti-human CD45RA, FITC</td><td>Miltenyi Biotec</td><td>130-092-247</td><td></td></tr><tr><td>anti-human CD45RO PE clone UCHL1</td><td>BD Biosciences</td><td>555493</td><td></td></tr><tr><td>anti-human CD4-PerCP clone SK3; mIgG1</td><td>BD Biosciences</td><td>345770</td><td></td></tr><tr><td>anti-human CD8-eFluor 450 (clone OKT8), mIgG2a</td><td>eBioscience</td><td>48-0086-42&amp;nbsp;</td><td></td></tr><tr><td>anti-human CTLA-4 (CD152), clone BNI3, mIgG2ak, Brilliant violet 421</td><td>BD Biosciences</td><td>562743</td><td></td></tr><tr><td>anti-human IFN-g FITC clone 4S.B3; mIgG1k</td><td>eBioscience</td><td>11-7319-81&amp;nbsp;</td><td></td></tr><tr><td>Brefeldin A-containing solution: GolgiPlug</td><td>BD Biosciences</td><td>555029</td><td></td></tr><tr><td>cDNA synthesis kit: SuperScript VILO(Reverse transcriptase) cDNA Synthesis Kit</td><td>Invitrogen</td><td>11754-250</td><td></td></tr><tr><td>Density centrifugation medium: Ficoll-Paque</td><td>GE healthcare</td><td>17-1440-03</td><td></td></tr><tr><td>DMSO 99.7%</td><td>Sigma Aldrich</td><td>D2650-5X5ML</td><td></td></tr><tr><td>FBS, heat inactivated</td><td>Invitrogen</td><td>10082-147</td><td></td></tr><tr><td>Fixable Viability Dye, eFluor 780&amp;nbsp;&amp;nbsp;</td><td>eBioscience</td><td>65-0865-14 or 65-0865-18&amp;nbsp;</td><td></td></tr><tr><td>Foxp3 Staining Buffer Set</td><td>eBioscience</td><td>00-5523-00</td><td>Caution: contains paraformaldehyde. Can be also bought in combined kit with antibody; 77-5774-40 Anti-Human Foxp3 Staining Set APC Clone: 236A/E7 Set</td></tr><tr><td>GlutaMAX (200 mM L-alanyl-L-glutamine)</td><td>Invitrogen</td><td>35050-061</td><td></td></tr><tr><td>human naive CD4 T cell isolation kit II</td><td>Miltenyi Biotec</td><td>130-094-131</td><td></td></tr><tr><td>Human serum albumin 50 g/L</td><td>Baxter</td><td>1501057</td><td></td></tr><tr><td>Ionomycin from Streptomyces conglobatus &gt;98%</td><td>Sigma Aldrich</td><td>I9657-1MG</td><td></td></tr><tr><td>MACS LS-columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>mouse IgG1 K Isotype Control APC Clone: P3.6.2.8.1</td><td>eBioscience</td><td>17-4714-42</td><td></td></tr><tr><td>mouse IgG1 K Isotype Control FITC 50 &amp;#956;g</td><td>eBioscience</td><td>11-4714-81&amp;nbsp;</td><td></td></tr><tr><td>mouse IgG2a isotype control, Brilliant violet 421, clone MOPC-173</td><td>BD Biosciences</td><td>563464</td><td></td></tr><tr><td>Pasteur pipet plastic, individually packed</td><td>Sarstedt</td><td>86.1172.001&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>PMA Phorbol 12-myristate 13-acetate</td><td>Sigma Aldrich</td><td>P1585-1MG&amp;nbsp;</td><td></td></tr><tr><td>Rapamycin</td><td>EMD (Merck)</td><td>553210-100UG</td><td></td></tr><tr><td>Recombinant Human IL-2, CF</td><td>R&amp;amp;D</td><td>202-IL-050/CF</td><td></td></tr><tr><td>Recombinant Human TGF-beta 1, CF</td><td>RnD</td><td>240-B-010/CF</td><td></td></tr><tr><td>RNA isolation kit: RNAqueous-Micro Kit</td><td>Ambion</td><td>AM1931&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>RPMI 1640 Medium&amp;nbsp;</td><td>Invitrogen</td><td>72400-054&amp;nbsp;</td><td></td></tr><tr><td>Sodium butyrate</td><td>Sigma Aldrich</td><td>B5887-250MG&amp;nbsp;</td><td></td></tr><tr><td>T cell culture medium: X-Vivo 15 medium, with gentamicin+phenolred</td><td>Lonza</td><td>04-418Q</td><td></td></tr><tr><td>TaqMan Gene Expression Assay, FOXP3 (Best Coverage)&amp;nbsp;</td><td>Applied Biosystems</td><td>4331182; assay ID: Hs01085834_m1</td><td>Caution: contains paraformaldehyde</td></tr><tr><td>TaqMan Gene Expression Assay, RPL13A (Best Coverage)</td><td>Applied Biosystems</td><td>4351370; assay ID: Hs04194366_g1&amp;nbsp;&amp;nbsp;</td><td>Caution: contains paraformaldehyde</td></tr><tr><td>TaqMan Gene Expression Master mix</td><td>Applied Biosystems</td><td>4369514</td><td></td></tr></tbody></table>

in vitro differentiation of human cd4+foxp3+ induced regulatory t cells (itregs) from na&#239;ve cd4+ t cells using a tgf-&#946;-containing protocol

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

Figure 1 shows a scheme of the experimental setup. Figure 2 shows a representative purity control staining for magnetically isolated na&#239;ve CD4+ T cells and nTregs.
Figure 3A shows the flow cytometry gating strategy and Figure 3B shows representative FOXP3 and CD25 flow cytometry stainings on day 6 of culture under the indicated iTreg or control c...

Watch this Scientific Journal Video about In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells iTregs from NaÃ¯ve CD4+ T Cells Using a TGF-Î²-containing Protocol at JoVE.com

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

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

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

in-vitro-differentiation-human-cd4-foxp3-induced-regulatory-t-cells

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JoVE Journal

Immunology and Infection

20.2K Views.  Karolinska Institutet, Karolinska University Hospital, & Science for Life Laboratory. This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from naïve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

Video: In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Naïve CD4+ T Cells Using a TGF-β-containing Protocol

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

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

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

Generation of Induced Regulatory T Cells from Primary Human Na&#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.

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

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

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 ...

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 ...

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

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

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

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

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

Generation of Human Regulatory T Cell Clones

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based on the identification of Treg through several activation-dependent cellular surface markers with varying expression levels in different physiologic and pathologic conditions. Populations isolated as &#8220;Treg&#8221; therefore often contain considerable numbers of non-Treg effector cells (i.e., Teff) which hamper the precise phenotypic and functional characterization of these cells, their genomic and proteomic characterization, their reliable enumeration in different states of health and disease, as well as their isolation and expansion for therapeutic purposes. The latter, in particular, remains a major hurdle, as the inadvertent expansion of effector cells homing in Treg-relevant cellular compartments (e.g., CD4+CD25+ T cells) may render Treg-based immunotherapy ineffective, or even harmful. This work presents a method that circumvents the problems associated with population-based isolation and expansion of Treg and shows that the generation of Treg candidate clones with the subsequent selection, culture, and expansion of only carefully vetted, monoclonal cells, enables the generation of an ultrapure Treg cell product that can be kept in culture for many months, enabling downstream investigation of these cells, including for possible therapeutic applications.

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based ...

Th17 Cell Differentiation from Naive CD4+ T-Cells Using Flow Cytometry

In Vitro Differentiation of Naive CD4+ T Cells into Pathogenic Th17 Cells in Mouse

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have been linked to a wide range of diseases in recent times, including multiple sclerosis (MS), rheumatoid arthritis, acute respiratory distress syndrome (ARDS), sepsis, and other autoimmune disorders. However, the currently known in vitro differentiation protocols have difficulty achieving high purity of pathogenic Th17 cells, with the induction efficiency often below 50%, which is a key challenge in in vitro experiments. In this protocol, we propose an enhanced in vitro culture and differentiation protocol for pathogenic Th17 cells, which is used to directly differentiate naive CD4+ T cells isolated from mouse spleens into pathogenic Th17 cells. This protocol provides detailed instructions on splenocyte isolation, purification of naive CD4+ T cells, and differentiation of pathogenic Th17 cells. Through this protocol, we can achieve a differentiation purity of approximately 90% for pathogenic Th17 cells, which meets the basic needs of many cellular experiments.

Author Spotlight: Achieving High-Purity In Vitro Differentiation of Th17 Cells Using Cytokine Concentration Modulation

This protocol describes the differentiation of na&#239;ve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from na&#239;ve CD4+ T cells using this differentiation method.

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...

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

To isolate the CD4-positive T cells, resuspend the white blood cell pellet in 5 milliliters of cell isolation buffer, and filter the cell suspension through a 30-micrometer strainer into a new 15-milliliter tube to remove any debris. After counting, resuspend the cells at a 1 x 107 cells per 90 microliters of buffer concentration, and add 10 microliters of anti-CD4 conjugated magnetic beads per 1 x 107 cells for a 15-minute incubation at 4 degrees Celsius, with mixing every 5 minutes.
While the cells are incubating, place a separation column onto a magnetic stand, and pre-wet the column with 2 milliliters of buffer. Then, wash the cells with 5 milliliters of buffer and resuspend the pellet in 1 milliliter of buffer. Load the sample onto the column, and collect the CD4-negative fraction in a 15-milliliter conical tube.
When all of the cells have passed through the column, wash the reservoir three times with 1 milliliter of buffer per wash before it dries. After the last wash, transfer the column to a new 15-milliliter conical tube, and plunge the column with 2 milliliters of cell isolation buffer to expel the positive bead fraction. Then, collect the CD4-positive cells by centrifugation.
For fluorescence-activated cell sorting of the na&#239;ve T cells, resuspend the bead-sorted CD4-positive cell pellet in 500 microliters of buffer, and mix the cells with an appropriate fluorescence-conjugated antibody cocktail. After 20 to 30 minutes on ice, protected from light, wash the cells in 5 milliliters of fresh buffer, and resuspend the pellet in 500 microliters of fresh buffer.
Filter the cells through a nylon mesh, and transfer the cells to a FACS tube pre-coded with 500 microliters of buffer. Gate to select the CD4-positive, CD25-negative, and CD44 low CD62 ligand high cell populations. Then, sort the na&#239;ve CD4-positive, CD25-negative, CD-44 low, CD-62 ligand high T cell fractions into a new 15-milliliter centrifuge tube and pellet the isolated na&#239;ve CD4-positive T cells by centrifugation.
For granulocyte-macrophage colony-stimulating factor, or GM-CSF, T helper cell differentiation, resuspend the sorted T cells at a 1 x 106 cells per milliliter concentration in complete RPMI medium containing 50 micromolar beta-mercaptoethanol, and aspirate the anti-CD3 epsilon antibody from each well of a pre-coded 48-well plate.
Then, seed 2.5 x 105 cells into each well along with IL-7, anti-CD28, and anti-IFN gamma, and incubate the cells at 37 degrees Celsius and 5% CO2 for three days. On the third day of culture, use a microscope to check the cell differentiation, and harvest the cells for centrifugation.
Resuspend the pellet in fresh complete RPMI medium for two washes, resuspending the cells in 1 milliliter of fresh complete medium after the second wash for counting. Split the cells into three equal volumes, and re-stimulate the first aliquot of cells with PMA and ionomycin in the presence of a protein transport inhibitor for 4 to 6 hours.
At the end of the stimulation, harvest the cells for staining with anti-CD4 antibody. After 30 minutes, fix the cells with 200 microliters of fixation buffer for 20 to 60 minutes, followed by two washes in 1 milliliter of permeabilization buffer per wash. Then, stain for intracellular cytokine expression for 30 minutes in the dark.

In Vitro Differentiation of Human CD4⁺FOXP3⁺ Induced Regulatory T Cells (iTregs) from Naïve CD4⁺ T Cells Using a TGF-β-containing Protocol

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Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

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