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.

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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...

Mit mehr als 34 Millionen Menschen mit HIV / AIDS weltweit schätzungsweise 2,5 Millionen Menschen im Jahr 2011 neu infiziert, die Notwendigkeit einer wirksamen HIV-Impfstoff, um die weltweite HIV-Epidemie einzudämmen bleibt von größter Bedeutung. Doch trotz drei Jahrzehnten intensiver Forschung haben sich die HIV-1-Vakzine Wirksamkeitsstudien bisher nur bescheidenen Schutz 1-3 führte und die Korrelate der schützenden Immunität bleiben weitgehend unverstanden. Erläuterung der Art der Immunantwort zum Schutz benötigt ist für die strategische Gestaltung einer wirksamen HIV-1-Impfstoff und anderen immuntherapeutischen Strategien gegen HIV-1-Infektion. Natürliche CD4 + regulatorische T-Zellen (Tregs) sind entscheidend für die Aufrechterhaltung der Homöostase Immunzellen durch Steuern übermäßige Aktivierung des Immunsystems und begrenzt somit immun-vermittelte Gewebeschädigung. Sie können aber auch zu unterdrücken Immunantworten gegen Pathogene und verhindern deren Beseitigung. Krebs und Hepatitis B Impfstoff-Studien haben gezeigt, dass die Verringerung der Aktivität von Tregs kann Impfstoff Reaktion und Antigen-spezifische Immunität gegen Viren 4-7 zu verbessern. In Zusammenhang mit der HIV-1-Infektion, ist die genaue Auswirkung von regulatorischen T-Zellen nicht vollständig verstanden. Tregs wurde gezeigt, dass die Virusreplikation in aktivierten T-Zellen 8 zu verringern und möglicherweise Auswirkungen auf Aktivierung des Immunsystems 9. Sie wurden auch gezeigt, dass HIV-1-spezifische Immunantworten, die negative Folgen für die Progression der Erkrankung haben könnten 10,11 unterdrücken. Somit bevor sie zu modulieren Treg Aktivität, um die Wirksamkeit eines HIV-1-Vakzine zu verbessern, ist es wichtig, weitere Einblicke in die Funktion gewinnen in diesem Zusammenhang Krankheit. Menschliche CD4 + regulatorische T-Zellen sind eine relativ knapp Zellpopulation, die etwa 5% der CD4 + T-Zellen im peripheren Blut und ihrer absoluten Zahlen weitere Abnahme mit HIV-assoziierten CD4 + T-Zell-Depletion 12 &lt;/ Sup&gt;. Diese Assays Treg Funktion, wie T-Zell-Proliferations-Assays mit Treg Co-Kultur zu beurteilen, mit relativ großer Zellzahlen 12. Daher kennzeichnenden Funktion und Spezifität von regulatorischen T-Zellen bei Patienten mit fortgeschrittener HIV-1-Erkrankung ist schwierig gewesen, trotz ihrer Bedeutung für die HIV-Pathogenese. Die ex vivo Isolation und Expansion von Tregs von HIV-1-Patienten könnte eine Lösung für einige dieser Einschränkungen zu überwinden. Hier beschreiben wir eine einfache und robuste Protokoll zu funktionellen Tregs von HIV-1-infizierten Individuen in vitro abgeleitet erweitern; wir weiter, wie man ihnen zu erklären, Phänotyp und testen ihre suppressive Funktion mit durchflusszytometrischen Assays. Wir glauben, dass dieses Protokoll wird der Zugang zu Tregs erleichtern und das Verständnis ihrer Rolle in HIV-1 Fortschreiten der Erkrankung.

<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 + regulatorischen T-Zellen (Tregs) sind potente Immunmodulatoren und haben eine wichtige Funktion in der menschlichen Immunhomöostase. Depletion von Tregs hat messbare Antigen-spezifischen T-Zell-Antworten in Impfstoff Einstellungen für Krebs und infektiösen Erregern führte. Jedoch bleibt ihre Rolle in HIV-1 Immuno-Pathogenese umstritten, da sie entweder könnte dazu dienen, schädliche HIV-1-assoziierten Aktivierung des Immunsystems zu unterdrücken und so langsam HIV-1 Fortschreiten der Krankheit zu unterdrücken oder auch HIV-1-spezifische Immunität zu fördern und dadurch Virus verbreiten. Understanding und modulierende Treg-Funktion im Rahmen der HIV-1 könnte zu möglichen neuen Strategien zur Immuntherapie oder HIV-Impfstoffe führen. Dennoch bleiben wichtige Fragen offen über ihre Rolle im Zusammenhang mit der HIV-1-Infektion, die sorgfältig untersucht werden muss. Stellvertretend für rund 5% des menschlichen CD4 + T-Zellen im peripheren Blut, das Studium der Treg Population hat sich als schwierig erwiesen, essondere in HIV-1-infizierten Individuen, wo HIV-1-assoziierten CD4 T-Zell-und damit Treg Erschöpfung auftritt. Die Charakterisierung von regulatorischen T-Zellen bei Patienten mit fortgeschrittener HIV-1 Erkrankung oder Gewebeproben, für die nur sehr kleinen biologischen Proben erhalten werden können, ist es äußerst schwierig. Wir schlagen vor, eine technische Lösung für diese Einschränkungen zu überwinden mit Isolierung und Expansion von Tregs von HIV-1-positiven Personen. Hier beschreiben wir eine einfache und robuste Methode erfolgreich ausbauen Tregs isoliert von HIV-1-Infizierten in vitro. Durchfluss-sortierten CD3 + CD4 + CD25 + CD127 niedrig Tregs wurden mit anti-CD3/anti-CD28 beschichtete Beads stimuliert und kultiviert in Gegenwart von IL-2. Die erweiterte Tregs exprimiert hohe FOXP3, CTLA4 und HELIOS im Vergleich zu herkömmlichen T-Zellen und erwiesen sich als sehr suppressive. Leichterer Zugang zu einer großen Anzahl von Tregs erlaubt Forschern, i Adressemportant Fragen über ihre Rolle im HIV-1 Immunpathogenese. Wir glauben, dass die Beantwortung dieser Fragen können nützliche Erkenntnisse für die Entwicklung eines wirksamen HIV-1-Impfstoff bereitzustellen.

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.

Neue Tools erweitern regulatorischen T-Zellen von HIV-1-infizierten Personen

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 Aufrufe.  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.