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...

HIV / AIDSの世界的な、新たに2011年に感染推定250万人と住んで34以上万人で、世界的なHIVの流行を抑制するための効果的なHIVワクチンの必要性が最優先のまま。しかし、強烈な研究努力の三十年にもかかわらず、これまでのHIV-1ワクチンの有効性試験は1-3のみささやかな保護をもたらしていると防御免疫の相関関係はよくわかっていないままです。保護のために必要とされる免疫応答の性質を解明することは効果的なHIV-1ワクチンとHIV-1感染症を標的と他の免疫療法戦略の戦略的設計のために不可欠です。 天然のCD4 +制御性T細胞（Tregの）このようにして、免疫媒介性組織損傷を制限する、過度の免疫活性化を制御することにより、免疫細胞の恒常性の維持に重要である。しかし、彼らはまた、病原体に対する免疫応答を抑制し、その隙間を防止することができる。がんとヘパティティ属Bワクチンの研究は、Tregの活性を低下させることは、ワクチン応答および4-7ウイルスに対する抗原特異的免疫を高めることができることを実証した。しかしながら、HIV-1感染の文脈において、制御性T細胞の正確な影響は完全には理解ままである。 Tregのは、活性化T細胞8およびおそらく影響免疫活性9にウイルス複製を減少させることが示された。彼らはまた、疾患の進行10,11のための否定的な結果を与える可能性がHIV-1特異的免疫応答を抑制することが示された。従って、HIV-1ワクチンの効力を高めるためにTregの活性を調節できるようになる前に、この病気の文脈でそれらの機能さらなる洞察を得るために重要である。 ヒトCD4 +制御性T細胞は、約5％のCD4 +末梢血中のT細胞、さらにその絶対数HIV-関連したCD4 + T細胞の枯渇12で減少し、相対的に少ない細胞集団である&lt;/&gt;（商標）。このような共培養をTreg細胞とT細胞増殖アッセイなどのTregの機能を評価するために、現在のアッセイは、12の比較的大きなセル番号を使用する。したがって、高度なHIV-1疾患を持つ個人で調節性T細胞の特徴的機能と特異性は、HIVの病因のために、その重要性にもかかわらず、挑戦されています。 HIV-1患者からex vivoで単離およびTregの拡大は、これらの制限のいくつかを克服するための解決策を表すことができる。ここでは、機能的なTregのin vitroで HIV-1感染患者由来拡大するための簡単かつ堅牢なプロトコルを記述し、我々は、さらにどのように表現型彼らに説明し、フローサイトメトリーアッセイを用いて、その抑制機能をテストします。私たちは、このプロトコルがTregのへのアクセスを容易にし、HIV-1疾患の進行で自分の役割を理解するのに役立つと信じています。

<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 +制御性T細胞（Tregの）は、強力な免疫調節因子であり、ヒト免疫ホメオスタシスにおいて重要な機能を果たす。 Tregの枯渇は、癌および感染性病原体ワクチンの設定の抗原特異的T細胞応答の測定可能増大するに至った。それらのいずれか有害なHIV-1に関連する免疫活性化を抑制する働きをするので、HIV-1疾患の進行を遅らせるか、あるいはHIV-1特異的免疫を抑制し、それによってウイルスを促進することができしかし、HIV-1免疫病因におけるそれらの役割は、議論の余地広がった。理解とHIV-1の文脈における変調Tregの機能は、免疫療法またはHIVワクチンのための潜在的な新しい戦略につながる可能性があります。しかし、重要な未解決の問題は慎重に検討する必要があり、HIV-1感染の文脈におけるその役割に残る。 、末梢血中のヒトCD4 + T細胞の約5％に相当するTregの人口が困難であることが証明されている勉強、エスpecially HIV-1関連のCD4 T細胞のHIV-1感染者やでそのTregの枯渇が発生します。ごく生物学的試料を得ることができる、高度なHIV-1疾患または組織サンプルは、極めて困難である。有する個体における調節T細胞の特徴付け我々は、HIV-1陽性の個体からTregの単離および拡張を使用して、これらの制限を克服するための技術的解決法を提案する。 ここで我々が正常にin vitroで HIV-1に感染した個体からTregの孤立を拡大するための、簡単かつ堅牢な方法について説明します。フローソートCD3 + CD4 + CD25 + CD127 低い Tregのanti-CD3/anti-CD28がコーティングされたビーズで刺激し、IL-2の存在下で培養した。拡張されたTregのは、FOXP3、高レベルの、CTLA4を表明し、HELIOS従来のT細胞と比較して、非常に抑制されることが示された。 Tregの多数に簡単にアクセスでは、研究者が私に対処できるようになりますHIV-1免疫病因における役割に関するmportant質問。我々は、これらの質問に答えると、効果的なHIV-1ワクチンの開発のための有益な洞察を提供するかもしれないと考えています。

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.

HIV-1に感染した個体からの制御性T細胞を増殖するための新しいツール

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 ビュー.  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.