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...

Dünyada HIV / AIDS ve yeni 2011 yılında enfekte yaklaşık 2,5 milyon kişi, dünya çapında HIV salgını engellemek için etkili bir HIV aşısı için ihtiyacı ile yaşayan fazla 34 milyon kişi ile her şeyden kalır. Ancak, yoğun araştırma çabalarının üç yıl olmasına rağmen, bugüne kadar HIV-1 aşı etkinliği çalışmalarda sadece mütevazı koruma 1-3 sonuçlandı ve koruyucu bağışıklık karşılıklarını tam olarak anlaşılamamıştır kalır. Koruma için gerekli bağışıklık tepkisinin doğasını açıklık bir HIV-1 aşı ve HIV-1 enfeksiyonu hedef alan diğer immünoterapötik stratejilerinin stratejik tasarımı için gereklidir. Doğal CD4 + düzenleyici T hücreleri (Tregs) dolayısıyla immün-aracılı doku hasarı sınırlama, aşırı immün aktivasyon kontrol ederek bağışıklık hücre homeostazının bakım için kritik öneme sahiptir. Ancak, onlar da patojenlere karşı bağışıklık yanıtlarını bastırmak ve boşluk önleyebilir. Kanser ve HEPAtitis B aşısı çalışmaları Tregs aktivitesini azaltarak aşı yanıtı ve virüslere karşı 4-7 antijen-spesifik bağışıklık geliştirmek olduğunu göstermiştir. Bununla birlikte, HIV-1 enfeksiyonu bağlamında, düzenleyici T hücrelerinin tam etkisini tam olarak anlaşılamamıştır. Tregs aktive edilmiş T hücreleri 8&#39;de virüs replikasyonu azaltmak ve muhtemelen immün sistemin 9 etkisi gösterilmiştir. Ayrıca, hastalık ilerlemesi 10,11 olumsuz sonuçlara sahip olabilir, HIV-1 spesifik bağışıklık tepkileri, bastırmak için gösterilmiştir. Bu nedenle, HIV-1 aşı etkinliğini artırmak için Treg aktivitesini modüle etmek mümkün edilmeden önce, bu hastalığın bağlamda fonksiyonu içine daha fazla fikir edinmek için önemlidir. İnsan CD4 + regülatör T hücreleri, yaklaşık 5 CD4 +% periferal kan T-hücrelerinde ve daha da kesin sayıları HIV ile ilişkili CD4 + T hücre tükenmesi 12 ile azalma temsil eden, nispeten az bir hücre popülasyonu olarak &lt;/&gt; Sup. Ko-kültür Treg ile böyle T hücresi çoğalma tahlilleri gibi Treg fonksiyonu değerlendirmek Mevcut analizler, göreceli olarak büyük bir hücre sayıları 12 kullanımı. Bu nedenle, gelişmiş olan kişilerde düzenleyici T hücrelerinin fonksiyon ve özgüllük karakterize HIV-1 hastalığı HIV patogenezinde önemi rağmen, zorlu olmuştur. HIV-1 hastalardan Tregs ex vivo izolasyonu ve genişleme bu sınırlamaları bazılarının üstesinden gelmek için bir çözüm temsil edebilir. Burada in vitro HIV-1 ile enfekte bireyler elde edilen fonksiyonel Tregs genişletmek için kolay ve güçlü bir protokol açıklar, biz daha fazla nasıl fenotip onlara açıklamak ve akım sitometrik deneyleri kullanarak baskılayıcı fonksiyonunu test edin. Biz bu protokolü Tregs erişimi kolaylaştırmak ve HIV-1 hastalığın ilerlemesine rolleri anlamak yardımcı olacağına inanıyoruz.

<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>
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		<tr>
			<td>Round-Bottom tube with 35 &mu;m a nylon mesh</td>
			<td>BD Falcon</td>
			<td>352235</td>
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		<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 + Düzenleyici T hücreleri (Tregs) güçlü bağışıklık modülatörleri ve insan bağışıklık dengesinin sağlanmasında önemli bir işlev görürler. Tregs tükenmesi kanser ve bulaşıcı patojen için aşı ortamlarda antijen-spesifik T hücre yanıtları ölçülebilir artışlara yol açmıştır. Ya da zararlı bir HIV-1 ile ilişkili immün aktivasyon bastırmak için vermektedir ve bu nedenle, HIV-1 hastalığın ilerlemesini yavaşlatmak ya da alternatif olarak, HIV-1 spesifik bağışıklık bastırmak ve böylece virüs teşvik olabilir Ancak, HİV-1 imüno-patogenezinde rol, tartışmalıdır yayıldı. Anlama ve HIV-1 bağlamında modüle Treg fonksiyonu immünoterapi veya HIV aşılar için potansiyel yeni stratejiler yol açabilir. Ancak, önemli açık sorular dikkatle incelenmesi gereken HIV-1 enfeksiyonu, bağlamında rolleri kalır. Treg popülasyonunda inceleyerek, insan CD4 + periferal kan T-hücrelerinin yaklaşık% 5&#39;ini temsil eden ES zor olduğu kanıtlanmıştırpecially HIV-1 ile ilişkili bir CD4 T hücresi HIV-1 ile enfekte olmuş kişilerde ve ile bu Treg tükenmesi oluşur. İleri HIV-1 hastalığı veya doku örnekleri, sadece çok küçük bir biyolojik numune elde edilebileceği için, bu nedenle son derece zorlu olan bireylerde, düzenleyici T hücrelerinin karakterizasyonu. Biz HIV-1 pozitif bireylerin Tregs izolasyonu ve genişleme kullanarak bu sınırlamaları aşmak için teknik bir çözüm öneriyoruz. Burada başarılı bir şekilde in vitro HIV-1 ile enfekte bireylerden Tregs izole genişletmek için kolay ve sağlam bir yöntem açıklanmaktadır. Akış kriteri CD3 + CD4 + CD25 + CD127 düşük Tregs anti-CD3/anti-CD28 ile kaplı boncuklar ile uyarılır ve IL-2 varlığında kültürlendi. Genişletilmiş Tregs FOXP3, yüksek düzeyde ifade edilir ve CTLA4 HELİOS geleneksel T-hücreleri ile karşılaştırıldığında ve son derece baskılayıcı olduğu gösterilmiştir. Tregs çok sayıda daha kolay erişim araştırmacılar i ele sağlayacakHIV-1 immünopatogenezinde rolleri ile ilgili mportant sorular. Biz bu sorulara cevap etkili bir HIV-1 aşısının geliştirilmesi için yararlı bilgiler sağlayabilir inanıyorum.

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.

Yeni Araçlar HIV-1 ile enfekte bireyler gelen Düzenleyici T hücreleri büyütmek için

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 Görüntüleme.  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.