This work was supported in part by research funding from the Elisabeth Glaser Pediatric AIDS Foundation (Pediatric HIV Vaccine Program Award MV-00-9-900-1429-0-00 to MMA), MGH/ECOR (Physician Scientist Development Award to MMA), NIH NIAID (KO8219 AI074405 and AI074405-03S1 to MMA), and the Harvard University Center for AIDS Research (CFAR), an NIH funded program (P30 AI060354) which is supported by the following NIH Co-Funding and Participating Institutes and Centers: NIAID, NCI, NICHD, NHLBI, NIDA, NIMH, NIA, FIC, and OAR. These studies were furthermore supported by the Bill &amp; Melinda Gates Foundation and the Terry and Susan Ragon Foundation.

1. Regulatory T cell isolation from HIV-1 Positive Blood
<ol>
	<li>Carefully transfer blood, collected in ACD tubes, into a 50 ml conical tube for a final volume of 15 ml blood per tube.</li>
	<li>Add 25 &mu;l/ml of blood of RosetteSep Human CD4+ T Cell Enrichment Cocktail, mix carefully and incubate 20 min at room temperature.</li>
	<li>Add 15 ml of PBS/2% FBS to the blood and mix carefully. Layer the diluted blood sample on top of 15 ml of Histopaque at room temperature in a 50 ml conical tube. Spin the conical tube for 20 min at 1,200 x g with a slow start and no brakes.</li>
	<li>Transfer the CD4+ T cell enriched PBMC layer in a new 50 ml conical tube, wash the cells by adding PBS/2% FBS and spin them down for 10 min at 1,200 x g. Then count the cells, wash again and resuspend the cells at about 20 x 106/200 &mu;l.</li>
	<li>Add the following antibodies (concentration): 
	anti-CD3-Phycoerythrin-Cyanine 7 (PE-Cy7) (1/100) 
	anti-CD4-Fluorescein Isothiocyanate (FITC) (1/40) 
	anti-CD25-Allophycocyanin (APC) (1/40) 
	anti-CD127-Phycoerythrin (PE) (1/20) 
	Incubate 30 min in the dark at 4 &deg;C</li>
	<li>Wash the cells with PBS/2% FBS. Resuspend the cells at 20 x 106/ml in PBS/2% FBS and filter them on a 35 &mu;m nylon mesh.</li>
	<li>Using a FACS Aria cell sorter equipped for handling biohazardous material, sort the CD3+CD4+CD25+CD127low Treg in X-VIVO 15 media (see gating strategy in Figure 1). Conventional T cells (CD3+CD4+CD25-CD127+) can be isolated and expanded as negative controls.</li>
</ol>
2. Cell Culture
<ol>
	<li>After isolation, wash the Treg with X-VIVO 15 media.</li>
	<li>Resuspend the cells at 250 x 103/ml in X-VIVO 15 media complemented with 10% Human Serum and Penicillin-Streptomycin (50 U/ml).</li>
	<li>Wash Human T-Activator CD3/CD28 beads according to manufacturer's protocol. Add beads to isolated Tregs at a ratio of 1:1 bead per cell.</li>
	<li>After two days of culture, double the media volume and add IL-2 (300 U/ml).</li>
	<li>Culture the Tregs for 2 weeks. Change media (X-VIVO 15/Human serum/P/S/IL-2) at days 5, 7, 9, 12. Add beads at a 1:1 ratio at day 9. When changing media, keep cells at 250 x 103/ml.</li>
</ol>
3. Phenotyping
At the end of the expansion culture, expanded CD3+CD4+CD25+CD127low Treg can be phenotyped by flow cytometry and compared to expanded CD3+CD4+CD25-CD127+ conventional T cells as a control.
<ol>
	<li>Harvest expanded Tregs/Tconvs and wash them in PBS. Label dead cells using the LIVE/DEAD Fixable Violet Dead Cell Stain Kit according to manufacturer's protocol. Wash the cells in PBS/2% FBS.</li>
	<li>Add the following antibodies (concentration): 
	anti-CD3-PECy7 (1/100) 
	anti-CD4-Qdot-655 (1/200) 
	anti-CD25-PECy5 (1/100) 
	Incubate 30 min in the dark at 4 &deg;C</li>
	<li>Wash the cells and perform the intracellular staining using the Foxp3/ Transcription Factor Staining Buffer Set according to manufacturer's protocol and the following antibodies: 
	anti-FOXP3-PE (1/50) 
	anti-HELIOS-FITC (1/40) 
	anti-CTLA4-APC (1/20) 
	Acquire the data on a flow cytometer.</li>
</ol>
4. Suppression Assay
At the end of the expansion culture, the suppressive function i.e. the capacity of the expanded Treg isolated from HIV-1 positive individuals to suppress the proliferation of activated T cells can be assessed in vitro.
<ol>
	<li>Thaw autologous cryopreserved ex vivo PBMCs. Leave them for about 3 hr in a 37 &deg;C incubator in RPMI 1640 medium containing penicillin/streptomycin, L-glutamine, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (=R+ media), and 10% FBS (=R10 media).</li>
	<li>Label the dead cells using the LIVE/DEAD Fixable Violet Dead Cell Stain Kit according to the manufacturer's protocol. Wash the cells in PBS/2% FBS.</li>
	<li>Incubate the cells with anti-CD3-PECy7 for 30 min in the dark at 4 &deg;C. Wash the cells with PBS/2% FBS. Resuspend the cells in PBS/2% FBS and filter them on a 35 &mu;m nylon mesh.</li>
	<li>Using a FACS Aria cell sorter equipped for handling biohazardous material, sort the viable CD3+ T cells in R10 media.</li>
	<li>Label the T cells with a cell tracing reagent such as CellTrace Violet or Vybrant CFDA SE Cell Tracer at 5 &mu;M diluted in PBS for 7 min at 37 &deg;C according to the manufacturer's protocol. Resuspend cells in R+ media supplemented with 10% human serum (=hR10 media) at 1 x 106/ml.</li>
	<li>Harvest the expanded Tregs, resuspend the cells at 0.5 x 106/ml in hR10 and prepare dilutions at 0.25 x 106/ml and 0.125 x 106/ml.</li>
	<li>Prepare anti-CD2/anti-CD3/anti-CD28 microbeads according to the manufacturer's protocol, resuspend the microbeads at 0.75 x 106/ml and prepare dilutions at 0.625, 0.562, 0.5 x 106/ml in hR10 media.</li>
	<li>In a 96 wells round bottom plate, transfer cells and beads according to the following plan:</li>
</ol>
<table border="1">
	<tbody>
		<tr>
			<td>T cells:Treg ratio</td>
			<td>1:0</td>
			<td>1:1/2</td>
			<td>1:1/4</td>
			<td>1:1/8</td>
		</tr>
		<tr>
			<td>T cells (50 &mu;l)</td>
			<td>1 x 106/ml</td>
			<td>1 x 106/ml</td>
			<td>1 x 106/ml</td>
			<td>1 x 106/ml</td>
		</tr>
		<tr>
			<td>Tregs (50 &mu;l)</td>
			<td>no</td>
			<td>0.5 x 106/ml</td>
			<td>0.25 x 106/ml</td>
			<td>0.125 x 106/ml</td>
		</tr>
		<tr>
			<td>Beads (100 &mu;l)</td>
			<td>0.5 x 106/ml</td>
			<td>0.75 x 106/ml</td>
			<td>0.625 x 106/ml</td>
			<td>0.562 x 106/ml</td>
		</tr>
		<tr>
			<td>hR10 (50 &mu;l)</td>
			<td>yes</td>
			<td>no</td>
			<td>no</td>
			<td>no</td>
		</tr>
		<tr>
			<td>i.e.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>T cells</td>
			<td>50 x 103</td>
			<td>50 x 103</td>
			<td>50 x 103</td>
			<td>50 x 103</td>
		</tr>
		<tr>
			<td>Tregs</td>
			<td>0</td>
			<td>25 x 103</td>
			<td>12.5 x 103</td>
			<td>6.25 x 103</td>
		</tr>
		<tr>
			<td>Beads</td>
			<td>50 x 103</td>
			<td>75 x 103</td>
			<td>62.5 x 103</td>
			<td>56.25 x 103</td>
		</tr>
	</tbody>
</table>
<ol start="9">
	<li>After 4 days of culture, wash the cells and incubate them for 30 min at 4 &deg;C with the following antibodies: 
	anti-CD3-PECy7 (1/100) 
	anti-CD4-APC (1/100) 
	anti-CD8-AF700 (1/100)</li>
</ol>
Acquire the data on a flow cytometer. Use the FlowJo proliferation platform to calculate the percentage of divided cells.

<ol>
	<li>Rerks-Ngarm, 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=Vaccination+with+ALVAC+and+AIDSVAX+to+prevent+HIV-1+infection+in+Thailand.">Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand.</a> N. Engl. J. Med. 361, 2209-2220 (2009).</li><li>Buchbinder, S. 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=Efficacy+assessment+of+a+cell-mediated+immunity+HIV-1+vaccine+(the+Step+Study):+a+double-blind,+randomised,+placebo-controlled,+test-of-concept+trial.">Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-concept trial.</a> Lancet. 372 (08), 1881-1893 (2008).</li><li>Pitisuttithum, 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=Randomized,+double-blind,+placebo-controlled+efficacy+trial+of+a+bivalent+recombinant+glycoprotein+120+HIV-1+vaccine+among+injection+drug+users+in+Bangkok,+Thailand.">Randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 vaccine among injection drug users in Bangkok, Thailand.</a> J. Infect. Dis. 194, 1661-1671 (2006).</li><li>Morse, M. 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=Depletion+of+human+regulatory+T+cells+specifically+enhances+antigen-specific+immune+responses+to+cancer+vaccines.">Depletion of human regulatory T cells specifically enhances antigen-specific immune responses to cancer vaccines.</a> Blood. 112, 610-618 (2008).</li><li>Furuichi, Y., 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=Depletion+of+CD25+CD4+T+cells+(Tregs)+enhances+the+HBV-specific+CD8++T+cell+response+primed+by+DNA+immunization.">Depletion of CD25+CD4+T cells (Tregs) enhances the HBV-specific CD8+ T cell response primed by DNA immunization.</a> World J. Gastroenterol. 11, 3772-3777 (2005).</li><li>Rech, A. J., Vonderheide, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+use+of+anti-CD25+antibody+daclizumab+to+enhance+immune+responses+to+tumor+antigen+vaccination+by+targeting+regulatory+T+cells.">Clinical use of anti-CD25 antibody daclizumab to enhance immune responses to tumor antigen vaccination by targeting regulatory T cells.</a> Ann. N.Y. Acad. Sci. 1174, 99-106 (2009).</li><li>Ruter, 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=Altering+regulatory+T+cell+function+in+cancer+immunotherapy:+a+novel+means+to+boost+the+efficacy+of+cancer+vaccines.">Altering regulatory T cell function in cancer immunotherapy: a novel means to boost the efficacy of cancer vaccines.</a> Front Biosci. 14, 1761-1770 (2009).</li><li>Moreno-Fernandez, M. E., Rueda, C. M., Rusie, L. K., Chougnet, C. A. <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+control+HIV+replication+in+activated+T+cells+through+a+cAMP-dependent+mechanism.">Regulatory T cells control HIV replication in activated T cells through a cAMP-dependent mechanism.</a> Blood. 117, 5372-5380 (2011).</li><li>Schulze Zur Wiesch, 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=Comprehensive+analysis+of+frequency+and+phenotype+of+T+regulatory+cells+in+HIV+infection:+CD39+expression+of+FoxP3++T+regulatory+cells+correlates+with+progressive+disease.">Comprehensive analysis of frequency and phenotype of T regulatory cells in HIV infection: CD39 expression of FoxP3+ T regulatory cells correlates with progressive disease.</a> J. Virol. 85, 1287-1297 (2011).</li><li>Kinter, 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=Suppression+of+HIV-specific+T+cell+activity+by+lymph+node+CD25++regulatory+T+cells+from+HIV-infected+individuals.">Suppression of HIV-specific T cell activity by lymph node CD25+ regulatory T cells from HIV-infected individuals.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 3390-3395 (2007).</li><li>Moreno-Fernandez, M. E., Presicce, P., Chougnet, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeostasis+and+function+of+regulatory+T+cells+in+HIV/SIV+infection.">Homeostasis and function of regulatory T cells in HIV/SIV infection.</a> J. Virol. , (2012).</li><li>Angin, 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=Preserved+Function+of+Regulatory+T+Cells+in+Chronic+HIV-1+Infection+Despite+Decreased+Numbers+in+Blood+and+Tissue.">Preserved Function of Regulatory T Cells in Chronic HIV-1 Infection Despite Decreased Numbers in Blood and Tissue.</a> J. Infect. Dis. 205, 1495-1500 (2012).</li><li>Seddiki, 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=Expression+of+interleukin+(IL)-2+and+IL-7+receptors+discriminates+between+human+regulatory+and+activated+T+cells.">Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells.</a> J Exp Med. 203, 1693-1700 (2006).</li><li>De Jager, P. 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=The+role+of+the+CD58+locus+in+multiple+sclerosis.">The role of the CD58 locus in multiple sclerosis.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 5264-5269 (2009).</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>Salomon, B., 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=B7/CD28+costimulation+is+essential+for+the+homeostasis+of+the+CD4+CD25++immunoregulatory+T+cells+that+control+autoimmune+diabetes.">B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes.</a> Immunity. 12, 431-440 (2000).</li><li>Malek, T. R., Bayer, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance,+not+immunity,+crucially+depends+on+IL-2.">Tolerance, not immunity, crucially depends on IL-2.</a> Nat. Rev. Immunol. 4, 665-674 (2004).</li><li>Hoffmann, P., Eder, R., Kunz-Schughart, L. A., Andreesen, R., Edinger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+in+vitro+expansion+of+polyclonal+human+CD4(+)CD25high+regulatory+T+cells.">Large-scale in vitro expansion of polyclonal human CD4(+)CD25high regulatory T cells.</a> Blood. 104, 895-903 (2004).</li><li>Putnam, A. 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=Expansion+of+human+regulatory+T-cells+from+patients+with+type+1+diabetes.">Expansion of human regulatory T-cells from patients with type 1 diabetes.</a> Diabetes. 58, 652-662 (2009).</li><li>Kreijveld, E., Koenen, H. J., Hilbrands, L. B., Joosten, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+expansion+of+human+CD4++CD25high+regulatory+T+cells+from+transplant+recipients+permits+functional+analysis+of+small+blood+samples.">Ex vivo expansion of human CD4+ CD25high regulatory T cells from transplant recipients permits functional analysis of small blood samples.</a> J. Immunol. Methods. 314, 103-113 (2006).</li><li>Ebinuma, 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=Identification+and+in+vitro+expansion+of+functional+antigen-specific+CD25++FoxP3++regulatory+T+cells+in+hepatitis+C+virus+infection.">Identification and in vitro expansion of functional antigen-specific CD25+ FoxP3+ regulatory T cells in hepatitis C virus infection.</a> J Virol. 82, 5043-5053 (2008).</li><li>Strauss, L., Czystowska, M., Szajnik, M., Mandapathil, M., Whiteside, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+responses+of+human+regulatory+T+cells+(Treg)+and+effector+T+cells+to+rapamycin.">Differential responses of human regulatory T cells (Treg) and effector T cells to rapamycin.</a> PLoS ONE. 4, e5994 (2009).</li><li>Heredia, 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=Rapamycin+causes+down-regulation+of+CCR5+and+accumulation+of+anti-HIV+beta-chemokines:+an+approach+to+suppress+R5+strains+of+HIV-1.">Rapamycin causes down-regulation of CCR5 and accumulation of anti-HIV beta-chemokines: an approach to suppress R5 strains of HIV-1.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 10411-10416 (1073).</li><li>Hoffmann, 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=Only+the+CD45RA++subpopulation+of+CD4+CD25high+T+cells+gives+rise+to+homogeneous+regulatory+T-cell+lines+upon+in+vitro+expansion.">Only the CD45RA+ subpopulation of CD4+CD25high T cells gives rise to homogeneous regulatory T-cell lines upon in vitro expansion.</a> Blood. 108, 4260-4267 (2006).</li><li>Hoffmann, 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=Loss+of+FOXP3+expression+in+natural+human+CD4+CD25++regulatory+T+cells+upon+repetitive+in+vitro+stimulation.">Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation.</a> Eur. J. Immunol. 39, 1088-1097 (2009).</li><li>Wang, J., Ioan-Facsinay, A., vander Voort, E. I., 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>Takahashi, 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=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> J. Exp. Med. 192, 303-310 (2000).</li><li>Thornton, A. 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=Expression+of+Helios,+an+Ikaros+transcription+factor+family+member,+differentiates+thymic-derived+from+peripherally+induced+Foxp3++T+regulatory+cells.">Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells.</a> J. Immunol. 184, 3433-3441 (2010).</li><li>Zheng, S. G., Gray, J. D., Ohtsuka, K., Yamagiwa, S., Horwitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+ex+vivo+of+TGF-beta-producing+regulatory+T+cells+from+CD4+CD25-+precursors.">Generation ex vivo of TGF-beta-producing regulatory T cells from CD4+CD25- precursors.</a> J. Immunol. 169, 4183-4189 (2002).</li><li>Gregori, S., Roncarolo, M. G., Bacchetta, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+in+vitro+generation+of+human+type+1+regulatory+T+cells.">Methods for in vitro generation of human type 1 regulatory T cells.</a> Methods Mol. Biol. 677, 31-46 (2011).</li></ol>

The authors declare that they have no competing financial interests.

Using the protocol described above, Tregs can be successfully isolated and expanded from HIV-1-infected individuals in vitro. Expanded Tregs express high levels of FOXP3, CTLA4 and HELIOS, are highly suppressive and display a highly demethylated Treg-Specific Demethylation Region (TSDR) locus of the FOXP3 gene (data not shown) 15, suggesting true origin from the regulatory T cell lineage, as opposed to activation-induced transient FOXP3 upregulation. Deep sequencing demonstrated that the TCR repertoir...

Con più di 34 milioni di persone che vivono con l&#39;HIV / AIDS in tutto il mondo e si stima che 2,5 milioni di persone recentemente infettate nel 2011, la necessità di un efficace vaccino HIV per frenare l&#39;epidemia di HIV in tutto il mondo rimane fondamentale. Tuttavia, nonostante tre decenni di intensi sforzi di ricerca, gli HIV-1 vaccino prove di efficacia fino ad oggi hanno portato solo una protezione modesta 1-3 ed i correlati di protezione immunitaria sono ancora limitate. Delucidare la natura della risposta immunitaria necessaria per la protezione è essenziale per la progettazione strategica di un efficace vaccino HIV-1 e di altre strategie immunoterapeutiche di targeting HIV-1. CD4 Naturali + cellule T regolatorie (Treg) sono fondamentali per il mantenimento dell&#39;omeostasi delle cellule immunitarie attraverso il controllo attivazione immunitaria eccessiva, limitando così i danni ai tessuti immuno-mediata. Tuttavia, possono anche sopprimere le risposte immunitarie contro gli agenti patogeni e prevenire la loro liquidazione. Cancro e Hepatitis studi vaccino B hanno dimostrato che diminuendo l&#39;attività di Treg può migliorare la risposta al vaccino e antigene-specifica immunità contro i virus 4-7. Tuttavia, nel contesto di infezione da HIV-1, l&#39;esatto impatto delle cellule T regolatorie rimane completamente compresa. Tregs hanno mostrato di diminuire la replicazione virale in cellule T attivate 8 ed eventualmente urtare attivazione immunitaria 9. Sono stati anche dimostrato di sopprimere le risposte immunitarie HIV-1-specifiche, che potrebbe avere conseguenze negative per la progressione della malattia 10,11. Così, prima di poter modulare l&#39;attività Treg per migliorare l&#39;efficacia di un vaccino di HIV-1, è importante ottenere una visione più la loro funzione in questo contesto malattia. CD4 + umane cellule T regolatorie sono una popolazione di cellule relativamente scarsa, che rappresentano circa il 5% delle cellule T CD4 + nel sangue periferico, e il loro numero assoluto ulteriore diminuzione con CD4 HIV-associati + T deplezione delle cellule 12 &lt;/ Sup&gt;. Saggi attuali per valutare la funzione Treg, come ad esempio T saggi di proliferazione delle cellule Treg con co-coltura, utilizzare il numero di cellule relativamente grandi 12. Pertanto, la funzione e la specificità delle cellule T regolatorie che caratterizza in persone con HIV in stadio avanzato 1 malattia è stato impegnativo, nonostante la loro importanza per la patogenesi dell&#39;HIV. L&#39;isolamento e l&#39;espansione ex vivo di Treg da HIV-1 i pazienti potrebbero rappresentare una soluzione per superare alcune di queste limitazioni. Qui si descrive un protocollo semplice e robusto per espandere Treg funzionale derivato da HIV-1 individui infettati in vitro, abbiamo ulteriormente spieghiamo come loro fenotipo e testare la loro funzione soppressiva utilizzando saggi di citometria a flusso. Crediamo che questo protocollo faciliterà l&#39;accesso al Treg e di aiuto per capire il loro ruolo in HIV-1 la progressione della malattia.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>RosetteSep Human CD4+ T Cell Enrichment Cocktail</td>
			<td>Stemcells technologies</td>
			<td>15062</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Histopaque</td>
			<td>Sigma</td>
			<td>H8889</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-CD3-PECy7</td>
			<td>BD Pharmingen</td>
			<td>557851</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-CD4-FITC</td>
			<td>eBioscience</td>
			<td>11-0049-42</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-CD25-APC</td>
			<td>eBioscience</td>
			<td>17-0259-42</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-CD127-PE</td>
			<td>BD Pharmingen</td>
			<td>557938</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Round-Bottom tube with 35 &mu;m a nylon mesh</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>X-VIVO 15</td>
			<td>Lonza</td>
			<td>04-418Q</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Mediatech</td>
			<td>30-001-Cl</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Human Serum</td>
			<td>Gemini Bio-Products</td>
			<td>100-512</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Human T-activator CD3/CD28</td>
			<td>Life Technologies</td>
			<td>111.31D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IL-2</td>
			<td>NIH Aids Research &amp; Reference Reagent Program</td>
			<td>136</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Violet Dead Cell Stain Kit</td>
			<td>Life technologies</td>
			<td>L34955</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-CD4-qdot-655</td>
			<td>Life Technologies</td>
			<td>Q10007</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-CD25-PECy5</td>
			<td>eBiosciences</td>
			<td>15-0259-42</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Foxp3 / Transcription Factor Staining Buffer Set</td>
			<td>eBiosciences</td>
			<td>00-5523-00</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-FOXP3-PE</td>
			<td>eBiosciences</td>
			<td>12-4776-42</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-HELIOS-FITC</td>
			<td>Biolegend</td>
			<td>137204</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-CTLA4-APC</td>
			<td>BD Pharmingen</td>
			<td>555855</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CellTrace Violet Cell Proliferation Kit</td>
			<td>Life Technologies</td>
			<td>C34557</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vybrant CFDA SE Cell Tracer Kit</td>
			<td>Life Technologies</td>
			<td>V12883</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Mediatech</td>
			<td>25-060-Cl</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Treg Suppression inspector</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-909</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-CD4-APC</td>
			<td>BD Pharmingen</td>
			<td>340443</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-CD8-AF700</td>
			<td>BD Pharmingen</td>
			<td>557945</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Sigma</td>
			<td>R0883</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Mediatech</td>
			<td>25-002-Cl</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>BD Vacutainer Blood Collection Tube w/ ACID CITRATE DEXTROSE (ACD)</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>364606</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FACSAria IIu Cell Sorter</td>
			<td>BD Biosciences</td>
			<td>-</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LSR II Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>-</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>Tree Star</td>
			<td>v887</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

new tools to expand regulatory t cells from hiv-1-infected individuals

CD4 + cellule T regolatorie (Treg) sono potenti modulatori immunitari e servono una funzione importante nella omeostasi immunitaria umana. L&#39;esaurimento delle Tregs ha determinato aumenti misurabili in antigene-specifiche risposte delle cellule T nelle impostazioni del vaccino per il cancro e gli agenti patogeni infettivi. Tuttavia, il loro ruolo in HIV-1 immuno-patogenesi rimane controversa, in quanto potrebbero servire sia per sopprimere deleteria attivazione immunitaria HIV-1-associata e quindi rallentare la progressione della malattia da HIV-1 o in alternativa sopprimere l&#39;immunità HIV-1-specifica e, quindi, promuovere virus diffondersi. La comprensione e la funzione Treg modulando nel contesto di HIV-1 potrebbero portare a potenziali nuove strategie di immunoterapia o vaccini contro l&#39;HIV. Tuttavia, importanti questioni aperte rimangono sul loro ruolo nel contesto di HIV-1, che deve essere studiato con attenzione. Rappresentando circa il 5% di CD4 + cellule T nel sangue periferico, studiando la popolazione Treg è dimostrato difficile, esspecialmente in HIV-1 individui infetti in cui l&#39;HIV-1-associati delle cellule T CD4 e si verifica che la deplezione Treg. La caratterizzazione delle cellule T regolatorie in individui con HIV in fase avanzata-1 malattia o campioni di tessuto, per cui solo molto piccoli campioni biologici possono essere ottenuti, è quindi estremamente impegnativo. Noi proponiamo una soluzione tecnica per superare queste limitazioni utilizzando l&#39;isolamento e l&#39;espansione delle Treg da individui HIV-1-positivi. Qui si descrive un metodo semplice e affidabile per espandere con successo Treg isolate da individui HIV-1-infetti in vitro. Flow-sorted CD3 + CD4 + CD25 + CD127 partire Tregs state stimolate con anti-CD3/anti-CD28 perline rivestite e coltivate in presenza di IL-2. L&#39;espanso Treg espresso alti livelli di FOXP3, CTLA4 e HELIOS rispetto alle cellule T convenzionali e risultano essere molto soppressiva. Più facile l&#39;accesso a un gran numero di Treg permetterà ai ricercatori di affrontare importante domande riguardanti il ​​loro ruolo in HIV-1 immunopatogenesi. Crediamo rispondere a queste domande può fornire indicazioni utili per lo sviluppo di un efficace vaccino HIV-1.

The expression of interleukin 2 receptor (CD25) and the interleukin 7 receptor (CD127) have been described as reliable surface markers to identify functional Treg populations 13 and have been shown to correlate with CD4+CD25+FOXP3+ Tregs 9,12. Figure 1 represents the gating strategy used to flow-sort single CD3+CD4+CD25+CD127low Tregs from PBMC isolated from an HIV-1-positive individual. The CD25/CD127 anti...

Watch this Scientific Journal Video about New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals at JoVE.com

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

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.

Nuovi strumenti per espandere cellule T regolatorie di individui HIV-1-infetti

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

new-tools-to-expand-regulatory-t-cells-from-hiv-1-infected-individuals

Research

JoVE Journal

Immunology and Infection

11.1K Views.  Ragon Institute of MGH, MIT, and Harvard. 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.