This work was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) grant (BB/H018573/1) and a BD Biosciences grant.

<ol>
	<li>Baumjohann, D., Ansel, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-mediated+regulation+of+T+helper+cell+differentiation+and+plasticity.">MicroRNA-mediated regulation of T helper cell differentiation and plasticity.</a> Nat Rev Immunol. 13 (9), 666-678 (2013).</li><li>Wilson, C. B., Rowell, E., Sekimata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+control+of+T-helper-cell+differentiation.">Epigenetic control of T-helper-cell differentiation.</a> Nat Rev Immunol. 9 (2), 91-105 (2009).</li><li>Pearce, E. L., Poffenberger, M. C., Chang, C. H., Jones, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fueling+immunity:+insights+into+metabolism+and+lymphocyte+function.">Fueling immunity: insights into metabolism and lymphocyte function.</a> Science. 342 (6155), 1242454 (2013).</li><li>Cherry, S. R., Biniszkiewicz, D., Van Parijs, L., Baltimore, D., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+Expression+in+Embryonic+Stem+Cells+and+Hematopoietic+Stem+Cells.">Retroviral Expression in Embryonic Stem Cells and Hematopoietic Stem Cells.</a> Mol and Cell Biol. 20 (20), 7419-7426 (2000).</li><li>Miller, A. D., Rosman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+retroviral+vectors+for+gene+transfer+and+expression.">Improved retroviral vectors for gene transfer and expression.</a> BioTechniques. 7 (9), 980-990 (1989).</li><li>Grez, M., Akg&#252;n, E., Hilberg, F., Ostertag, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+virus,+a+recombinant+murine+retrovirus+with+expression+in+embryonic+stem+cells.">Embryonic stem cell virus, a recombinant murine retrovirus with expression in embryonic stem cells.</a> Proc Nat Acad Sci USA. 87 (23), 9202-9206 (1990).</li><li>Naviaux, R. K., Costanzi, E., Haas, M., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pCL+vector+system:+rapid+production+of+helper-free,+high-titer,+recombinant+retroviruses.">The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses.</a> J Virol. 70 (8), 5701-5705 (1996).</li><li>Filipowicz, W., Bhattacharyya, S. N., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+post-transcriptional+regulation+by+microRNAs:+are+the+answers+in+sight?.">Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight?.</a> Nat Rev Genetics. 9 (2), 102-114 (2008).</li><li>He, L., Hannon, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+small+RNAs+with+a+big+role+in+gene+regulation.">MicroRNAs: small RNAs with a big role in gene regulation.</a> Nat Rev Genetics. 5 (7), 522-531 (2004).</li><li>Bernstein, E., Kim, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dicer+is+essential+for+mouse+development.">Dicer is essential for mouse development.</a> Nat Genetics. 35 (3), 215-217 (2003).</li><li>Cobb, B. S., Hertweck, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+Dicer+in+immune+regulation.">A role for Dicer in immune regulation.</a> J Exp Med. 203 (11), 2519-2527 (2006).</li><li>Chong, M. M. W., Rasmussen, J. P., Rudensky, A. Y., Rundensky, A. Y., Littman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNAseIII+enzyme+Drosha+is+critical+in+T+cells+for+preventing+lethal+inflammatory+disease.">The RNAseIII enzyme Drosha is critical in T cells for preventing lethal inflammatory disease.</a> J Exp Med. 205 (9), 2005-2017 (2008).</li><li>Liston, A., Lu, L. F., O'Carroll, D., Tarakhovsky, A., Rudensky, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dicer-dependent+microRNA+pathway+safeguards+regulatory+T+cell+function.">Dicer-dependent microRNA pathway safeguards regulatory T cell function.</a> J Exp Med. 205 (9), 1993-2004 (2008).</li><li>Zhou, X., Jeker, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+miRNA+disruption+in+T+reg+cells+leads+to+uncontrolled+autoimmunity.">Selective miRNA disruption in T reg cells leads to uncontrolled autoimmunity.</a> J Exp Med. 205 (9), 1983-1991 (2008).</li><li>Singh, Y., Garden, O. A., Lang, F., Cobb, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-15b/16+Enhances+the+Induction+of+Regulatory+T+Cells+by+Regulating+the+Expression+of+Rictor+and+mTOR.">MicroRNA-15b/16 Enhances the Induction of Regulatory T Cells by Regulating the Expression of Rictor and mTOR.</a> J Immunol. 195 (12), 5667-5677 (2015).</li><li>Sauer, S., Bruno, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+signaling+controls+Foxp3+expression+via+PI3K,+Akt,+and+mTOR.">T cell receptor signaling controls Foxp3 expression via PI3K, Akt, and mTOR.</a> Proc Nat Acad Sci USA. 105 (22), 7797-7802 (2008).</li><li>Yagi, J., Arimura, Y., Takatori, H., Nakajima, H., Iwamoto, I., Uchiyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+influences+Th+cell+differentiation+by+controlling+the+capacity+for+IL-2-induced+IL-4+production+by+naive+CD4++T+cells.">Genetic background influences Th cell differentiation by controlling the capacity for IL-2-induced IL-4 production by naive CD4+ T cells.</a> Int Immunol. 18 (12), 1681-1690 (2006).</li><li>Singh, Y., Garden, O. A., Lang, F., Cobb, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+regulate+T-cell+production+of+interleukin-9+and+identify+hypoxia-inducible+factor-2a+as+an+important+regulator+of+T+helper+9+and+regulatory+T-cell+differentiation.">MicroRNAs regulate T-cell production of interleukin-9 and identify hypoxia-inducible factor-2a as an important regulator of T helper 9 and regulatory T-cell differentiation.</a> Immunology. 149, 74-86 (2016).</li></ol>

The authors have no conflicting interest in the publication of this work.

이들 발전과 기능은 종종 키 조정기의 발현에 의해 결정되는 바와 같이 레트로 바이러스 매개 된 유전자의 과발현은 헬퍼 T 세포의 기능을 분석하기위한 강력한 방법이다. 크게 내인성 유전자들 위에서 발현 여러 이슈를 도입 할 수 있기 때문에, 결과의 해석에주의가 필요하다. 따라서,이 기술은 함수의 관련성을 확인하기 위해 다른 조합한다. 예를 들어, 과발현이 가능한 경우 siRNA를 또는 유전자 녹아웃을 사용하여 단축 된 표현에 의해 보완되어야한다. 의 miRNAs, 우리는 miRNA의 15 경쟁력 억제제 행동 사이트를 대상으로 인공 miRNA의를 과발현 바이러스를 사용하여 차단하는 것과 과발현 실험을 보완. 레트로 바이러스 형질 도입 된 세포는 RNA 및 단백질 분석을 포함하는 생화학 적 분석에 이용 될 수있다. 그러나,이 실험의 주요 제한은 입술 전달 효율은형질 도입 및 untransduced 세포의 혼합 인구 ulting. 따라서 이러한 분석은 가능성이 가장 높은 GFP + 인구의 정렬이 필요합니다. 마지막으로, 체외 분화 분석은 생체 내 실험과 결합하고, 이것이 달성 될 수있는 한 가지 방법은 적응 적 마우스로 형질 도입 된 T 세포의 전사 및 분화 및 면역 반응에 미치는 영향에 따라이다한다. 이 시스템의 주요 한계들 중 하나는 레트로 바이러스 캡시드 내로 패키징 될 수있는 RNA 게놈의 크기이다. 우리의 경험에서, 좋은 바이러스 생산을 제공 MIG의 레트로 바이러스 시스템의 최대 삽입 크기는 3-3.5 KB입니다. 이들은 불량 바이러스 역가를 제공 따라서 큰 유전자는이 시스템을 해석 할 수 없다. 이 시스템의 유전자 연구의 다양한 유용하므로 그러나, 대부분의 유전자가이 크기보다 작다. 레트로 바이러스 전달이 protoco 내에서 여러 가지 대안으로LS가 사용되어왔다. 많은 연구자 안정적 레트로 바이러스 유전자 (예 16 참조) 발현 패키징 세포주를 이용했다. 그러나, 우리는 PCL 에코 헬퍼 바이러스 벡터의 공동 형질 표준의 HEK 293T 세포를 사용하여 최고 역가를 얻었다. 나이브 헬퍼 T 세포의 분리는 자성 비드와 세포 분리 컬럼 프로토콜 대신 정렬 셀을 통해 달성 될 수 있지만, 이는 셀 소터에 대한 액세스를 요구하고, 일종의 시간에 대한 비용은 일반적으로 비드 시약보다 더 높다. 마지막으로, 서로 다른 서브 세트에 헬퍼 T 세포를 구별하는 데 사용되는 작동 조건에 변동이있다. 예를 들어, 너무 오래 세포의 TCR 자극은 TREG 유도 조건에 노출하기 전에 유도 (16)을 억제 할 수 있습니다. 레트로 바이러스 발현 세포의 자극에 의해 유도되는 세포의 분열을 필요로하기 때문에 문제가 될 수있다. 그럼에도 불구하고, 우리는 O / N의 액티브이 프로토콜을 사용하여 효율적인 TREG 유도를 발견레트로 바이러스 전달하기 전에 ATION. 이러한 프로토콜 내에서 성공적인 응용 프로그램은 몇 가지 요소가 필요합니다. 높은 역가 레트로 바이러스 제제는 중요하다 HEK의 293T 세포 때문에 높은 품질의 DNA와 정확하게 준비 배 HBS의 효율적인 형질 전환이 필요합니다. 형질 감염된 DNA를 잘 발현은 세포가 너무 희박한 또는 조밀 한 경우 세포가 활발히 성장하고,이 억제되도록 요구하기 때문에 또한 HEK 293T 세포의 세포 밀도는 형질의 시점에서 약 50 %가 될 필요가있다. 형질 전환시 최적의 농도에서 세포는 바이러스 수집 단계 동안 어떤 시점에서 합류에 도달해야하지만, 그들은 모든 방법 마지막 컬렉션을 통해 높은 역가의 바이러스 주식을 생산하는 것입니다. 헬퍼 T 세포의 효율적인 분화 세포 좋은 품질이되도록 분리 된 세포는도 1에 도시 된 순도되도록 요구한다. 마찬가지로, 셀의 품질은 쥐 FR에 의존그들은 고립 된 엄마. 이러한 연구를 위해, 우리 6-8 주령 C57BL / 6 마우스를 사용하고 있습니다. 이전 마우스는 덜 순진 세포를 가질 수 있고, 다른 균주는 차별화 다를 수 있습니다. T 세포는 TH2 반응을 유도하기 어려울 수 있으며, 상기 C57BL / 6 바와 같이 예를 들어, BALB / c 마우스 그래서 C57BL / 6 마우스 17보다 TH2 반응에 더 쉽다. 또한, 분화 조건들은 실험실 실험실에서 약간 다를 수 있고, 다양한 편광 상태의 사이토 카인 농도는 적정 될 필요가있을 수 있으므로 유전자 과발현의 효과는 서브 - 최적의 조건에서 명백해질 수있다. 마지막으로, 세포 증식의 과발현 유전자의 영향이나 편광 상태는 관심의 유전자의 측정 이렇게 효과 편광 시약의 타이밍 및 농도를 최적화 필요할 수 전달 효율에 영향을 미칠 수있다. 이러한 모든 요소를 ​​최적화하는 것은이 시스템에 유익한 결과를 가져올 것이다.

The immune response must be highly regulated to eliminate infections but prevent attacks on self-tissue that lead to autoimmunity. Helper T cells play an essential role in regulating the immune response, and a great deal of effort has been undertaken to understand their development and function (illustrated in several recent reviews 1-3). However, many questions remain, and many approaches have been utilized to study the mechanisms controlling helper T cell development and function. These have ranged from the use of in vitro cell culture systems to whole animals. Cell culture systems, especially those using cell lines, offer the benefit of ease of use and the ability to generate large amount of material to do sophisticated biochemical analyses. However, they suffer from their limited ability to reproduce the actual conditions occurring in an immune response. In contrast, whole animal experiments offer the benefit of relevance, but they can suffer from difficulties in manipulation and the ability to perform precise controls in addition to their large costs and ethical implications. Nevertheless, the vast majority of helper T cells studies today still require the use of whole animal experiments involving primary T cells because of the inability of cell lines to duplicate the exact steps occurring in the whole animal. Therefore, it is essential to utilize cost effective approaches that are highly informative. 
Genetics is one powerful tool to study helper T cell development and function, yet traditional methods involving gene knockouts or transgenes are time consuming and expensive so they are often out of reach of small labs. However, retroviral transduction offers a powerful, rapid and, cost effective genetic approach to study the mechanisms of specific gene products. Therefore, it is commonly used in papers studying helper T cell development and function. 
We have optimized a procedure for retroviral transduction of helper T cells. It utilizes the pMIG (Murine stem cell virus-Internal ribosomal entry site-Green fluorescent protein) retroviral expression vector, in which the gene of interest can be cloned and thereby expressed from the retrovirus long terminal repeat (LTR) 4. In addition, downstream of the inserted gene of interest is an internal ribosome entry sequence (IRES) followed by the green fluorescent protein (GFP) gene so transduced cells can easily be followed by their expression of GFP. The vector was originally derived from the Murine Stem Cell Virus (MSCV) vectors, which contain mutations in repressor binding sites in the LTRs making them resistant to silencing and thus, giving high expression in many cell types including helper T cells 5,6. Production of high titer retrovirus requires a simple transient transfection protocol of human embryonic kidney (HEK) 293T cells with the MIG vector and a helper virus vector that expresses the retroviral GAG, Pol, and Env genes. For this the pCL-Eco helper virus vector 7 works well in producing high titer replication incompetent retroviruses. 
Here these protocols for retroviral production and transduction of primary murine T cells are described in addition to some of our results using this approach to study miRNA regulation of gene expression controlling helper T cell differentiation. miRNAs are small RNAs of approximately 22 nucleotides in length that post-transcriptionally regulate gene expression by targeting homologous sequences in protein encoding messenger RNAs and suppressing translation and inducing message instability 8,9. miRNAs play critical roles in developmental gene regulation. They are essential in the earliest stages of development, as embryos that cannot produce miRNAs die at a very early stage 10. In addition miRNAs are important later on in the development of many tissues. They are thought to function by fine-tuning the expression of genes required for developmental programs 1. In helper T cells miRNAs play multiple roles and are required for regulatory T cell (Treg) development 11-14. We used retroviral transduction as a means to dissect the mechanisms of miRNA regulation of Treg differentiation 15. Through such studies important individual miRNAs were determined by retroviral-mediated overexpression. Subsequently, relevant genes regulated by these miRNAs were identified in order to understand the molecular pathways regulated by miRNAs in helper T cell differentiation.

<table border="0" cellpadding="0" cellspacing="0" width="763">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">RPMI</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">R8758</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">DMEM</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">D5671</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Penicillin Streptomycin solution</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">L-Glutamine</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">&#946;-mercaptoethanol</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">M3148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">DPBS</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">MIG vector</td>
			<td style="width:143px;">Addgene</td>
			<td style="width:167px;">Plasmid 9094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">pCL-Eco vector</td>
			<td style="width:143px;">Addgene</td>
			<td style="width:167px;">Plasmid 12371</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Cell strainer</td>
			<td style="width:143px;">BD Falcon</td>
			<td style="width:167px;">352350</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Magnetic beads mouse CD4 cell kit&#160;</td>
			<td style="width:143px;">Invitrogen (Dynabeads)</td>
			<td style="width:167px;">11415D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Beads&#160;</td>
			<td style="width:143px;">Miltenyi Biotech</td>
			<td style="width:167px;">130-048-102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">MS cell separation columns</td>
			<td style="width:143px;">Miltenyi Biotech</td>
			<td style="width:167px;">130-042-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">LS cell separation columns</td>
			<td style="width:143px;">Miltenyi Biotech</td>
			<td style="width:167px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">CD25 Biotenylated MAb&#160;</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:167px;">85059</td>
			<td>clone 7D4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">CD62L Biotenylated MAb&#160;</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:167px;">553149</td>
			<td>clone MEL-14</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Polybrene (Hexadimethrine Bromide)</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">107689</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Anti-CD3</td>
			<td style="width:143px;">eBiosciences</td>
			<td>16-0031-85</td>
			<td>clone 145-2C11&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Anti-CD28</td>
			<td style="width:143px;">eBiosciences</td>
			<td>16-0281-85</td>
			<td>clone 37.51&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Anti-IL-4</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:167px;">559062</td>
			<td>clone 11B11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Anti-IFN-gamma</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:167px;">559065</td>
			<td>clone XMG1.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Anti-IL-2</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:167px;">554425</td>
			<td>cloneJES6-5H4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Recombinant IL-12 p70</td>
			<td style="width:143px;">eBiosciences</td>
			<td style="width:167px;">14-8121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Recombinant IL-4</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:167px;">550067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Recombinant TGF-beta</td>
			<td style="width:143px;">eBiosciences</td>
			<td>14-8342-62</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Recombinant IL-6</td>
			<td style="width:143px;">eBiosciences</td>
			<td style="width:167px;">14-8061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Recombinant IL-2</td>
			<td style="width:143px;">eBiosciences</td>
			<td style="width:167px;">14-8021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">PMA&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">P8139</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Ionomycin</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">I0634</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Brefeldin A</td>
			<td style="width:143px;">eBiosciences</td>
			<td style="width:167px;">00-4506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Paraformaldehyde</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">16005</td>
			<td>Paraformaldehyde is toxic so use appropriate caution when handling&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Foxp3 staining buffer set</td>
			<td style="width:143px;">eBiosciences</td>
			<td style="width:167px;">00-5523</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Anti-CD4&#160; FITC</td>
			<td style="width:143px;">eBiosciences</td>
			<td style="width:167px;">11-0041</td>
			<td>clone GK1.5&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Anti-CD8a perCP-cy5.5</td>
			<td style="width:143px;">eBiosciences</td>
			<td>45-0081-80</td>
			<td>clone 53-6.7&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Anti-MHCII PE</td>
			<td style="width:143px;">eBiosciences</td>
			<td style="width:167px;">12-0920</td>
			<td>&#160;clone HIS19</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Anti-CD25 PE</td>
			<td style="width:143px;">eBiosciences</td>
			<td>12-0251-82</td>
			<td>&#160;clone PC61.5&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Anti-CD62L PE</td>
			<td style="width:143px;">eBiosciences</td>
			<td>12-0621-82</td>
			<td>clone MEL-14&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Anti-CD44 APC</td>
			<td style="width:143px;">eBiosciences</td>
			<td style="width:167px;">17-0441</td>
			<td>clone IM7&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Anti-IFN-gamma FITC&#160;</td>
			<td style="width:143px;">eBiosciences</td>
			<td>11-7311-81</td>
			<td>clone XMG1.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Anti-IL-4 PE</td>
			<td style="width:143px;">BD Biosciences</td>
			<td>554435</td>
			<td>clone 11B11</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:287px;">Anti-IL-9 PE or APC</td>
			<td style="width:143px;">eBiosciences/Biolegend</td>
			<td>50-8091-82/514104</td>
			<td>clone RM9A4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Anti-IL-17a PE</td>
			<td style="width:143px;">BD Biosciences</td>
			<td>559502</td>
			<td>clone TC11-18H10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Anti-Foxp3&#160; APC or PE</td>
			<td style="width:143px;">eBiosciences</td>
			<td>17-5773-82/12-5773-80&#160;</td>
			<td>clone FJK-16s</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">P9333</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2HPO4-2H2O</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">71643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextrose/Glucose</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">G7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES, free acid</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">H3375</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NH4Cl</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">A9434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disodium EDTA</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">D2900000</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KHCO3</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">237205</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">&#160;CaCl2&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">C5670</td>
			<td></td>
		</tr>
	</tbody>
</table>

retroviral transduction of helper t cells as a genetic approach to study mechanisms controlling their differentiation and function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

이 실험 시스템의 성공은 T 세포와 고역가 레트로 바이러스 제제의 고순도 집단을 필요로한다. 대표적인 결과가 성공적인 실험 예로서 여기에 도시되어있다.도 1은도 2. 순진한 헬퍼 T 세포 격리 프로토콜의 각 단계에서 전후 선택된 집단의 전형적인 순도를 보여주고도 3은 GFP 발현을 레트로 바이러스 생산의 분석을 도시 형질 감염된 HEK의 293T 세포 (그림 2)와 형질 도입 T 세포 (그림 3). HEK 293T 세포의 형질 전환 효율은 다른 레트로 바이러스 작 제물에 따라 크게 변할 수 있지만, 이것은 종종 GFP + T 세포의 수를 관찰 레트로 바이러스 생산의 수준과 상관 없다. 또한, GFP + T 세포의 숫자는 편광 상태에 따라 달라질 수있다. 또한, mGFP의 발현 수준 EAN 일체화 바이러스 매수에 따라 달라질 수있는 삽입 유전자의 전사에 통합 사이트 효과 및 바이러스 전 사체에 영향을주는 전사 후 조절 기전. 마지막으로, 그림 4의 miRNAs은 miR-15B는 / (16)가 과발현 때 우리는 헬퍼 T 세포 분화 관찰 한 전형적인 결과를 보여줍니다. 이러한 결과 때문에 진정한 효과 헬퍼 T 세포의 다양한 제제를 사용하여 여러 반복 실험의 통계적 분석에 의해 입증되어야 개별 실험에서 발생할 수있는 변화들을 보여준다. 이 실험에서, TH2 반응은 한 Th1 반응하는 경향이 있기 때문에 여기에 사용 된 C57BL / 6 라인 관찰하기가 어려울 수있다. 유사하게, IL-9 염색 배경 위에서 검출하기 어려울 수있다. 따라서, 아이소 타입 컨트롤을하고 올바른 조지아을 보장하기 위해 적절한 보상을 설정하는 데 필수적이다사이토 카인의 발현 팅. 우리의 결과에서 우리는은 miR-15B / 16 구성 요소 Rictor과 mTOR의 15의 발현을 억제을 통해 신호 전달 경로에서 mTOR를 억제함으로써 iTreg 유도을 향상 것으로 나타났습니다. 미르 15B / 16 때때로 개별 실험 TH0,받은 Th1 및 Th17 분화에 영향을 미칠 수 있지만, 다수의 반복 실험에서 시험 큰 영향은 없다. 반면은 miR-15B / 16 과발현이 크게 TH9 차별화를 (참조 18 참조) 억제 않습니다. <img alt="그림 1" src="/files/ftp_upload/54698/54698fig1.jpg" /> 그림 분리의 각 단계에서 헬퍼 T 세포의 1. 일반적인 순도. 표시된 항원의 결과 세포 계측법 대표 흐름은 앞으로 분산 형 (FSC)와 사이드 분산 형 (SSC) 플롯에서 지정한 살아있는 세포의 게이트에서 표시됩니다. (A) 사전 및 사후 CD4 음의 선택. CD4의 발현 프로파일,CD8a 및 MHCII이 표시됩니다. 이러한 헬퍼 T 세포의 보충 및 세포 독성 T 세포의 손실 및 MHC 클래스 II 발현 세포를 나타낸다. 좋은 정화는이 단계에서 90 % CD4 + T 세포 ~ 초래한다. (B) CD25를 선택합니다. 왼쪽에서 CD4, CD8a 및 MHCII의 발현 프로파일, 그리고 오른쪽에있는 CD4이며 CD25 발현 사전 및 사후 선택을 프로필. CD25 부정적인 선택한 셀이 점&gt; 95 %에서 CD4 + CD25해야한다 -. (C) CD62L보세요. CD4, CD8a 및 MHCII 발현 프로파일은 왼쪽에 표시됩니다. 오른쪽에 CD62L과 CD44의 발현 양상은 포스트 선택된 셀의 CD4와 CD62L 식 프로필과 함께 사전 및 사후 CD62L 선택된 셀에 표시됩니다. CD62L 선택되면 거의 모든 메모리 셀 (CD44 +)를 10~15% 이펙터 세포 (CD62L 저)를 함유 나이브 헬퍼 T 세포의 고농축 인구 이탈 제거된다. 모든특정 매개 변수에 대한 FACS 프로필, 동등한 설정 및 저울에 걸쳐 유지 하였다. 숫자는 게이트 집단 내 세포의 비율을 나타낸다. 초기 선택 후 세포의 크기에 약간의 감소는 프로토콜 동안 기계적 스트레스에 아마도 때문이다. <a href="http://ecsource.jove.com/files/ftp_upload/54698/54698fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/54698/54698fig2.jpg" /> 도 레트로 바이러스 형질 감염된 HEK 293T 세포에 2. 분석. GFP 발현은 형질 감염되지 않은 또는 형질 감염 및 바이러스 배양 상청액 수집 한 후 분석 하였다 하나의 HEK 293T 세포에 나타낸다. GFP 분석은 첫 번째 패널에서 금융위원회에 게이트와 SSC 플롯에서 살아있는 세포에서 수행되었다. 숫자는 게이트 영역 내의 GFP + 세포의 비율을 나타낸다. 일반적인 transfection 효율이 30~90% 사이의 범위. <a href="http://ecsource.jove.com/files/ftp_upload/54698/54698fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/54698/54698fig3.jpg" /> 도 레트로 바이러스 형질 헬퍼 T 세포 3. 분석. GFP 발현 TH0,받은 Th1, TH2, TH9, Th17에서 분화 후 레트로 바이러스 형질 도입 헬퍼 T 세포에 도시 한 3 일간 TREG 편광 상태이다. 분석은 FSC / SSC 패널에 표시된 라이브 및 활성화 세포에 문이되었다. 전달 효율은 구조 및 편광 상태에 따라 10~75%에 따라 다를 수 있습니다. 마찬가지로, GFP 발현의 평균 형광 강도는 다릅니다. 수있는 <a href="http://ecsource.jove.com/files/ftp_upload/54698/54698fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 그림은 miR-(15b)의 4 효과 / 다른 편광 상태에서 헬퍼 T 세포 분화에 16 과발현. 대표적인 사이토 카인 프로필이 GFP + 그림 3에서 세포의 인구에 표시됩니다. <a href="http://ecsource.jove.com/files/ftp_upload/54698/54698fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="1 번 테이블" src="/files/ftp_upload/54698/54698table1.jpg" /> 표 1 :.이 프로토콜에 사용되는 버퍼 <a href="http://ecsource.jove.com/files/ftp_upload/54698/54986table1.xlsx" target="_blank">Excel 스프레드 시트로이 표를 다운로드하려면 여기를 클릭하십시오.</a> <table fo:keep-together.within-page="1"><tbody><tr><td colspan="3"> 돕는 사람T 세포 편광 상태 </td></tr><tr><td></td><td></td><td></td></tr><tr><td> TH0 </td><td> 항 IL-4 </td><td> 5 μg의 / ㎖ </td></tr><tr><td></td><td> 안티 IFN-γ </td><td> 5 μg의 / ㎖ </td></tr><tr><td></td><td></td><td></td></tr><tr><td> TH1 </td><td> 재조합 IL-12 </td><td> 20 ng를 / ㎖ </td></tr><tr><td></td><td> 항 IL-4 </td><td> 5 μg의 / ㎖ </td></tr><tr><td></td><td></td><td></td></tr><tr><td> TH2 </td><td> 재조합 IL-4 </td><td> 각 40ng / ㎖ </td></tr><tr><td></td><td> 안티 IFN-γ </td><td> 5 μg의 / ㎖ </td></tr><tr><td></td><td></td><td></td></tr><tr><td> TH9 </td><td> 재조합 TGF-β </td><td> 2.5 ng를 / ㎖ </td></tr><tr><td></td><td> 재조합 IL-4 </td><td> 각 40ng / ㎖ </td></tr><tr><td></td><td> 안티 IFN-γ </td><td> 10 μg의 / ㎖ </td></tr><tr><td></td><td></td><td></td></tr><tr><td> Th17 </td><td> 재조합 TGF-β </td><td> 2.5 ng를 / ㎖ </td></tr><tr><td></td><td> 재조합 IL-6 </td><td> 50 ng를 / ㎖ </td></tr><tr><td></td><td> 안티 IFN-γ </td><td> 5 μg의 / ㎖ </td></tr><tr><td></td><td> 항 IL-4 </td><td> 5 μg의 / ㎖ </td></tr><tr><td></td><td> 항 IL-2 </td><td> 5 μg의 / ㎖ </td></tr><tr><td></td><td></td><td></td></tr><tr><td> Tregs </td><td> 재조합 TGF-β </td><td> 2.5 ng를 / ㎖ </td></tr><tr><td></td><td> 재조합 IL-2 </td><td> 5 NG / ㎖ </td></tr></tbody></table> 표 2 : 헬퍼 T 세포 서브 세트의 편광 상태.

Watch this Scientific Journal Video about Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function at JoVE.com

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

많은 실험 시스템은 면역 반응의 T 세포의 발달 및 기능을 조절하는 메카니즘을 이해하는 데에 이용되고있다. 여기 레트로 바이러스 형질 도입을 이용하여 유전 적 접근 경로를 규정하는 식별 경제적 시간 효율적이고, 무엇보다도 매우 유익하다 설명한다.

공부하는 유전 적 접근으로 헬퍼 T 세포의 레트로 바이러스 형질 도입은 메커니즘 자신의 차별화 및 기능 제어

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

retroviral-transduction-helper-t-cells-as-genetic-approach-to-study

Research

JoVE Journal

Immunology and Infection

10.9K 조회수.  The Royal Veterinary College.  많은 실험 시스템은 면역 반응의 T 세포의 발달 및 기능을 조절하는 메카니즘을 이해하는 데에 이용되고있다. 여기 레트로 바이러스 형질 도입을 이용하여 유전 적 접근 경로를 규정하는 식별 경제적 시간 효율적이고, 무엇보다도 매우 유익하다 설명한다. 

비디오: Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

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

마우스 T - 세포에서 T - 세포 수용체의 Retroviral 전달

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 ...

연구

면역학 및 감염병학

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

세균성 접착 연구에 전단 응력을 소개합니다

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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.

T Lymphocytes쪽으로 유도 Pluripotent 줄기 세포의 분화 감독

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

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

<em&gt; 체외</em인간 대식 세포의 분화와 양극화의 이질성을 연구&gt; 모델

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

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.

나이브 CD4의 아데노 바이러스 형질은 Treg의 분화를 연구하는 세포를 T

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 ...

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

레이디 얼 이동성 및 레트로 바이러스 복제 벡터 형질 도입, 비 접착 Alloresponsive T 림프구의 세포 독성 기능

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

골수 전구 세포의 레트로 바이러스 형질 도입은 T 세포 수용체 Retrogenic 마우스를 생성하는

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

생성<em&gt; 드 노보</em&gt; 중심 Hemichain의 레트로 바이러스 형질 도입에 의해 항원 특이 적 인간 T 세포 수용체

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

생체 외에서 마우스 Granulocyte 대 식 세포 콜로 니 자극 인자 (GM-CSF)-T (THGM) 도우미 셀 생산

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.