Nina Nagel is gratefully acknowledged for technical assistance during the video shoot and experimental preparation. We thank Eva-Maria Weiss for help with the intracellular FOXP3 staining protocol and Elisabeth Suri-Payer and Nina Oberle for establishing the nTreg isolation protocol. Matilda Eriksson and Peri Noori are acknowledged for laboratory management. 
Funding: A.S. was supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme, the Dr. &#197;ke Olsson Foundation and KI research foundations; A.S. and J.T. were supported by a CERIC (Center of Excellence for Research on Inflammation and Cardiovascular disease) grant, J.T. was supported by Vetenskapsr&#229;det Medicine and Health (Dnr 2011-3264), Torsten S&#246;derberg Foundation, FP7 STATegra, AFA Insurance and Stockholm County Council.

<ol>
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The authors have nothing to disclose.

所描述的协议使人类CD4 + FOXP3 +来自人幼稚CD4 + T细胞的iTregs的鲁棒诱导。它包括我们最近描述的，使用TGF-β，全反式维甲酸和雷帕霉素的组合，对于具有优异的体外抑制功能22的iTregs的感应一个新的协议。相比其他发表的方案，另一个优点是不同iTreg种群的并联感应由不同的协议，从而使的某些iTreg诱导因子效应的直接比较，与在存在单独的IL-2活化的对照细胞沿着。所描述的...

CD4 + CD25 + FOXP3 +调节性T细胞（Treg细胞）抑制其他免疫细胞，并外周耐受的关键介质，防止自身免疫和炎症反应过度1。 Treg细胞的重要性是由人类疾病immunodysregulation多内分泌腺病肠病X连锁综合征（IPEX），其中的Treg的损失，由于在`master&#39; Treg细胞转录因子叉头框P3突变（FOXP3）例举导致严重的全身性自身免疫性疾病，致命自幼。然而，调节性T细胞作为免疫系统，因为他们也可以妨碍某些设置2抗肿瘤免疫力一把双刃剑。调节性T细胞的数量和功能的治疗操纵因此受到众多临床研究。在癌症中，调节性T细胞耗竭可能是可取的，临床的方法取得了一些成功鼓励进一步研究3。在自身免疫和炎性疾病，除了在塞弗Treg细胞的治疗效果拉尔小鼠疾病模型，继Treg细胞转移的最近最早在人的试验，以防止移植物抗 -host病（GVHD）4 - 7和评估安全性在治疗1型糖尿病8显示了非常有前途的结果。 天然存在的Tregs（nTregs）包括在保持健康9胸腺衍生tTregs和外周诱导的pTregs，具有非冗余必要功能- 11。然而，nTreg数量有限，鼓励诱导调节性T细胞（iTregs的） 在体外从幼稚T细胞前体12的补充方法。仍然稳定的iTregs的，大概是由于缺乏在FOXP3基因座13的所谓的Treg特异性去甲基化区域（TSDR）去甲基化，仍令人担忧和若干研究表明， 在诱导的Tregs 体内更稳定14。 迄今为止，FOXP3仍然是最好的M蛋白arker对于调节性T细胞，但也并非绝对特定，因为人类的常规的CD4 + CD25-T细胞中瞬时激活后15,16表达FOXP3的中间水平。虽然显著进展已经阐明FOXP3表达的调节而完成的，仍有许多关于特别是在人类细胞中FOXP3的诱导，稳定性和功能被发现。尽管差异nTregs， 在体外诱导FOXP3 + CD4 + T细胞可以作为一个模型系统以研究FOXP3诱导分子机制和作为起点到在将来开发的协议，其允许产生的iTregs的那些更类似于体内产生的Treg，这可能是适用于在将来继转移策略。 没有`金standard&#39;协议以诱导人的iTregs，和当前的协议都基于模仿Treg细胞诱导条件在体内被开发:白细胞介素-2（IL-2）和转化生长因子β（TGF-β）信号对于FOXP3诱导体内 17是至关重要的，和全反式维甲酸（ATRA） -这是在体内通过肠相关树突细胞产生的-被经常用来增强FOXP3感应在体外 18 - 21。我们已经开发出利用丁酸22中 ，最近显示，以增加鼠Treg的感应23,24一个肠道微生物衍生的短链脂肪酸的其他人Treg诱导协议。我们最近还建立用于产生具有体外优异的抑制功能的iTregs的一个新的协议，通过使用TGF-β，ATRA的组合和雷帕霉素22，后者是雷帕霉素（mTOR的）的临床批准哺乳动物靶抑制剂是已知促进人Treg扩张25,26中FOXP3维护。 该方法描述了在再现的体外代人CD4 + FOXP3 +使用一组不同的条件的iTregs，并通过流式细胞术和定量逆转录聚合酶链反应及其后续的表型的（QRT-PCR），以揭示FOXP3的表达等的Treg签名分子如的协议特定的图案如CD25，CTLA-4，EOS，以及IFN-γ和SATB1 表达 22的压制。所产生的细胞群可用于关于抑制活性官能测定或对分子研究，无论是关于一般的FOXP3调节剂或研究特定的某些化合物，如丁酸盐或雷帕霉素的效果。驾驶Treg细胞分化的分子机制进一步的了解是在自身免疫或癌症的未来治疗方法专门针对参与调节性T细胞的产生和功能分子高度相关。

<table border="0" cellpadding="0" cellspacing="0" width="1689">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:21px;width:501px;">All-trans retinoic acid</td>
			<td style="width:176px;">Sigma Aldrich</td>
			<td style="width:248px;">R2625-50MG&#160;&#160;</td>
			<td style="width:764px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anit-human Foxp3-APC clone 236A/E7</td>
			<td>eBioscience</td>
			<td>17-4777-42</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anti-human CD25 microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-983</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anti-human CD25-PE</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anti-human CD28 antibody, LEAF Purified&#160;</td>
			<td>Biolegend</td>
			<td>302914</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anti-human CD3 Antibody, LEAF Purified&#160;</td>
			<td>Biolegend</td>
			<td>317315</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:501px;">anti-human CD45RA , FITC</td>
			<td>Miltenyi Biotec</td>
			<td style="width:248px;">130-092-247</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anti-human CD45RO PE clone UCHL1</td>
			<td>BD Biosciences</td>
			<td>555493</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anti-human CD4-PerCP clone SK3; mIgG1</td>
			<td>BD Biosciences</td>
			<td>345770</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anti-human CD8-eFluor 450 (clone OKT8), mIgG2a</td>
			<td>eBioscience</td>
			<td>48-0086-42&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:501px;">anti-human CTLA-4 (CD152), clone BNI3, mIgG2ak, Brilliant violet 421</td>
			<td>BD Biosciences</td>
			<td>562743</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anti-human IFN-g FITC clone 4S.B3; mIgG1k</td>
			<td>eBioscience</td>
			<td>11-7319-81&#160;</td>
			<td style="width:764px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:501px;">Brefeldin A-containing solution: GolgiPlug</td>
			<td>BD Biosciences</td>
			<td>555029</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:501px;">cDNA synthesis kit: SuperScript VILO(Reverse transcriptase) cDNA Synthesis Kit</td>
			<td style="width:176px;">Invitrogen</td>
			<td style="width:248px;">11754-250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:501px;">Density centrifugation medium: Ficoll-Paque</td>
			<td style="width:176px;">GE healthcare</td>
			<td style="width:248px;">17-1440-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;">DMSO 99,7%</td>
			<td>Sigma Aldrich</td>
			<td>D2650-5X5ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS, heat inactivated</td>
			<td>Invitrogen</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Fixable Viability Dye, eFluor 780&#160;&#160;</td>
			<td>eBioscience</td>
			<td>65-0865-14 or 65-0865-18&#160;</td>
			<td style="width:764px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">Foxp3 Staining Buffer Set</td>
			<td>eBioscience</td>
			<td>00-5523-00&#160;</td>
			<td style="width:764px;">Caution, contains Paraformaldehyde 
			Can be also bought in combined kit with antibody; 77-5774-40 Anti-Human Foxp3 Staining Set APC Clone: 236A/E7 Set&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">GlutaMAX (200 mM L-alanyl-L-glutamine)</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">human naive CD4 T cell isolation kit II</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-131</td>
			<td></td>
		</tr>
		<tr height="20">
			<td align="left" height="20" style="height:21px;"><img alt="nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT" height="1" src="file:///C:/Users/DAVIDK~1/AppData/Local/Temp/msohtmlclip1/01/clip_image002.png" width="2" />
			<table cellpadding="0" cellspacing="0">
				<tbody>
					<tr>
						<td height="20" style="height:21px;width:501px;">human naive CD4 T cell isolation kit II</td>
					</tr>
				</tbody>
			</table>
			</td>
			<td align="left"><img alt="nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT" height="1" src="file:///C:/Users/DAVIDK~1/AppData/Local/Temp/msohtmlclip1/01/clip_image004.png" width="1" />
			<table cellpadding="0" cellspacing="0">
				<tbody>
					<tr>
						<td height="20" style="height:21px;width:176px;">Miltenyi Biotec</td>
					</tr>
				</tbody>
			</table>
			</td>
			<td>130-094-131</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Human serum albumin 50 g/l</td>
			<td>Baxter</td>
			<td>1501057</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Ionomycin from Streptomyces conglobatus &#62;98%</td>
			<td style="width:176px;">Sigma Aldrich</td>
			<td>I9657-1MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">MACS LS-columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:501px;">mouse IgG1 K Isotype Control APC Clone: P3.6.2.8.1&#160;</td>
			<td>eBioscience</td>
			<td>17-4714-42&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">mouse IgG1 K Isotype Control FITC 50 ug&#160;</td>
			<td>eBioscience</td>
			<td>11-4714-81&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:501px;">mouse IgG2a isotype control, Brilliant violet 421, clone MOPC-173</td>
			<td>BD Biosciences</td>
			<td>563464</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Pasteur pipet plastic, individually packed</td>
			<td>Sarstedt</td>
			<td>86.1172.001&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">PMA PHORBOL 12-MYRISTATE 13-ACETATE</td>
			<td>Sigma Aldrich</td>
			<td>P1585-1MG&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Rapamycin</td>
			<td>EMD (Merck)</td>
			<td>553210-100UG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Recombinant Human IL-2, CF</td>
			<td>R&#38;D</td>
			<td>202-IL-050/CF</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:501px;">Recombinant Human TGF-beta 1, CF</td>
			<td>RnD</td>
			<td>240-B-010/CF</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">RNA isolation kit: RNAqueous-Micro Kit</td>
			<td style="width:176px;">Ambion</td>
			<td>AM1931&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">RPMI 1640 Medium&#160;</td>
			<td>Invitrogen</td>
			<td>72400-054&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Sodium butyrate</td>
			<td>Sigma Aldrich</td>
			<td>B5887-250MG&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">T cell culture medium: X-Vivo 15 medium, with gentamicin+phenolred</td>
			<td>Lonza</td>
			<td>04-418Q</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">TaqMan Gene Expression Assay, FOXP3 (Best Coverage)&#160;</td>
			<td style="width:176px;">Applied Biosystems</td>
			<td style="width:248px;">4331182; assay ID: Hs01085834_m1</td>
			<td>Caution, contains Paraformaldehyde</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">TaqMan Gene Expression Assay, RPL13A (Best Coverage)</td>
			<td style="width:176px;">Applied Biosystems</td>
			<td>4351370; assay ID: Hs04194366_g1&#160;&#160;</td>
			<td>Caution, contains Paraformaldehyde</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:501px;">TaqMan Gene Expression Master mix</td>
			<td style="width:176px;">Applied Biosystems</td>
			<td style="width:248px;">4369514</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

图1示出实验装置的方案。 图2示出了用于磁性分离的幼稚的CD4 + T细胞和nTregs的代表性纯度对照染色。 ˚Figure 3A显示仪设门的流动和图3B显示代表FOXP3和CD25流式细胞仪染色的培养6日表示iTreg或控制的条件下。 在体外刺激后，大多数细胞上调CD25，它是在雷帕霉素?...

Watch this Scientific Journal Video about In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells iTregs from NaÃ¯ve CD4+ T Cells Using a TGF-Î²-containing Protocol at JoVE.com

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

本协议描述从幼稚CD4 + T细胞在体外可重复产生和人为调节性T细胞（iTregs的）的表型。不同的协议FOXP3诱导允许与各自的协议获取特定表型iTreg的研究。

在从幼稚的CD4 + T细胞的人CD4 + FOXP3 +诱导调节性T细胞（iTregs的）使用含有TGF-β-协议的体外分化

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

in-vitro-differentiation-human-cd4-foxp3-induced-regulatory-t-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

20.1K 次观看.  Karolinska Institutet, Karolinska University Hospital, & Science for Life Laboratory. 本协议描述从幼稚CD4 + T细胞在体外可重复产生和人为调节性T细胞（iTregs的）的表型。不同的协议FOXP3诱导允许与各自的协议获取特定表型iTreg的研究。 

视频: In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Naïve CD4+ T Cells Using a TGF-β-containing Protocol

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

T细胞受体在小鼠T细胞的逆转录病毒转导

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 ...

科研

免疫与感染

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.

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

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

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

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.

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.

新工具展开调节性T细胞的HIV-1感染的个体

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

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.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

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.

对T细胞推导<em&gt;在体外</em&gt;从小鼠胚胎干细胞

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

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

在从幼稚的CD4 ⁺ T细胞^的人CD4 ⁺ FOXP3 ⁺诱导调节性T细胞（iTregs的）使用含有TGF-β-协议的体外分化

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