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 t...

<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>

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...

With more than 34 million individuals living with HIV/AIDS worldwide and an estimated 2.5 million people newly infected in 2011, the need for an effective HIV vaccine to curb the worldwide HIV epidemic remains paramount. However, despite three decades of intense research efforts, the HIV-1 vaccine efficacy trials to date have resulted in only modest protection 1-3 and the correlates of protective immunity remain poorly understood. Elucidating the nature of the immune response needed for protection is essential for the strategic design of an effective HIV-1 vaccine and other immunotherapeutic strategies targeting HIV-1 infection.
Natural CD4+ regulatory T cells (Tregs) are critical to the maintenance of immune cell homeostasis by controlling excessive immune activation, thus limiting immune-mediated tissue damage. However, they can also suppress immune responses against pathogens and prevent their clearance. Cancer and Hepatitis B vaccine studies have demonstrated that decreasing the activity of Tregs can enhance vaccine response and antigen-specific immunity against viruses 4-7. However, in the context of HIV-1 infection, the exact impact of regulatory T cells remains incompletely understood. Tregs were shown to decrease virus replication in activated T cells 8 and possibly impact immune activation 9. They were also shown to suppress HIV-1-specific immune responses, which could have negative outcomes for disease progression 10,11. Thus, before being able to modulate Treg activity to enhance the efficacy of an HIV-1 vaccine, it is important to gain further insight into their function in this disease context.
Human CD4+ regulatory T cells are a relatively scarce cell population, representing about 5% of CD4+ T cells in the peripheral blood, and their absolute numbers further decrease with HIV-associated CD4+ T cell depletion 12. Current assays to assess Treg function, such as T cell proliferation assays with Treg co-culture, use relatively large cell numbers 12. Therefore, characterizing function and specificity of regulatory T cells in individuals with advanced HIV-1 disease has been challenging, despite their importance for HIV pathogenesis.
The ex vivo isolation and expansion of Tregs from HIV-1 patients could represent a solution to overcome some of these limitations. Here we describe an easy and robust protocol to expand functional Tregs derived from HIV-1 infected individuals in vitro; we further explain how to phenotype them and test their suppressive function using flow cytometric assays. We believe this protocol will facilitate access to Tregs and help understanding their role in HIV-1 disease progression.

<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>
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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.

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.

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 visualizações.  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.