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.

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

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

マウスT細胞におけるT細胞受容体のレトロウイルス導入

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

免疫・感染学

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

ナチュラルキラー細胞傷害性アッセイにおけるHIV - 1感染プライマリCD4 + T細胞を標的細胞としての準備と使用

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

初代ヒトナイーブおよびメモリーT細胞から誘導した調節性T細胞の生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1感染CD4の生得センシングを評価<sup&gt; +</sup&gt;使用して形質細胞様樹状細胞によるT細胞<em&gt;エクスビボ</em&gt;共培養系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

T細胞の導出<em&gt;体外</em&gt;マウス胚性幹細胞からの

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T細胞は、樹状細胞からTransinfectionによって細菌をキャプチャ

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

表現型および慢性リンパ球性脈絡髄膜炎ウイルスに感染したマウスから単離された活性化制御性T細胞の機能解析

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

自己免疫に対する幹細胞由来の抗原特異的制御性T細胞の開発

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.

HIV-1感染細胞からのエキソソームのSILACベースのプロテオミクス特性評価

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.