新工具展开调节性T细胞的HIV-1感染的个体
Summary
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.
Abstract
CD4 +调节性T细胞（Treg细胞）是强有力的免疫调节剂，在人体免疫稳态中发挥重要作用。导致可测量的增加，在癌症和感染病原体的疫苗设置的抗原特异性T细胞应答的调节性T细胞的耗竭。然而，它们的作用在HIV-1免疫发病机制仍存在争议，因为它们既可以起到抑制有害的HIV-1相关的免疫激活，从而减慢HIV-1疾病的进展或者抑制HIV-1的特异性免疫力，从而促进病毒蔓延。理解和调节Treg细胞功能的背景下，HIV-1可能导致免疫治疗或艾滋病毒疫苗的潜在的新战略。然而，重要的开放问题依然存在的背景下，HIV-1的感染，这需要仔细研究自己的角色。
占大约5％的人类CD4 + T细胞在外周血中，研究调节性T细胞的人口已被证明是困难的，第ESpecially在HIV-1感染个体的HIV-1相关的CD4 T细胞和调节性T细胞耗竭与发生。调节性T细胞在个体与先进的HIV-1病或组织样品，其中只有非常小的生物样品可以通过以下方式获得，因此极具挑战性的表征。我们提出了一个技术解决方案来克服这些限制，使用调节性T细胞的分离和扩增HIV-1阳性的个体。
在这里，我们介绍一个简单而可靠的方法，成功地扩大调节性T细胞体外 HIV-1 感染者隔离。流的排序条件CD3 + CD4 + CD25 + CD127 低调节性T细胞刺激与anti-CD3/anti-CD28包被的磁珠，并在IL-2存在下培养。扩大调节性T细胞表达高水平的FOXP3，CTLA4 HELIOS相比传统的T细胞被证明是高度抑制。更容易获得大量的调节性T细胞，将使研究人员能够解决我的mportant问题，他们在HIV-1免疫病理的作用。我们认为，回答这些问题可能为一个有效的HIV-1疫苗的发展提供有益的启示。
Introduction
拥有超过3400万个人生活与艾滋病毒/艾滋病在世界范围，估计有250万人新感染在2011年，需要一个有效的HIV疫苗，以遏制全球艾滋病疫情仍然是最重要的。然而，尽管深入研究三十年的努力，已导致HIV-1疫苗的有效性试验只有适度保护1-3保护性免疫的相关性仍然知之甚少。阐明保护所需的免疫反应的性质是必不可少的一种有效的HIV-1的针对HIV-1感染的疫苗和免疫治疗策略的策略设计。
天然的CD4 +调节性T细胞（Treg细胞）通过控制过度的免疫激活，从而限制了免疫介导的组织损伤的免疫细胞稳态的维持是至关重要的。然而，他们也可以对病原体的抑制免疫反应，并防止其间隙。巨蟹座和HEPAtitis疫苗的研究表明，调节性T细胞活性的降低可以提高疫苗的反应和抗原特异性的抗病毒4-7。然而，调节性T细胞在HIV-1感染的背景下，确切的影响还不完全清楚。调节性T细胞减少8活化的T细胞和病毒复制的可能影响免疫激活9。他们还抑制HIV-1的特异性免疫反应，这可能会产生负面的结果病情恶化10,11。因此，能够调节的调节性T细胞活性的HIV-1疫苗，以增强疗效之前，重要的是进一步深入了解这种疾病的上下文中它们的功能。
人CD4 +调节性T细胞是一种相对稀缺的细胞群体，占约5％的CD4 + T细胞的外周血中，和他们的绝对数量进一步减少与艾滋病毒相关的CD4 + T细胞耗竭12 </ SUP>。电流检测，以评估调节性T细胞的功能，如T细胞增殖实验与调节性T细胞共培养，使用比较大的手机号码12。因此，表征个人与先进的功能和特异性的调节性T细胞在HIV-1疾病已具有挑战性的，尽管他们的艾滋病毒发病机制中的重要性。
从HIV-1患者调节性T细胞体外分离和扩展，可以代表一个解决方案来克服这些限制。在这里，我们介绍一个简单而强大的协议扩大功能的调节性T细胞的HIV-1 感染者体外来自我们进一步解释了表型他们和测试使用流式细胞仪检测其抑制功能。我们相信，这项协议将Tregs的便利和帮助了解自己的角色在HIV-1疾病的进展。
Protocol
Representative Results
Discussion
Using the protocol described above, Tregs can be successfully isolated and expanded from HIV-1-infected individuals in vitro. Expanded Tregs express high levels of FOXP3, CTLA4 and HELIOS, are highly suppressive and display a highly demethylated Treg-Specific Demethylation Region (TSDR) locus of the FOXP3 gene (data not shown) 15, suggesting true origin from the regulatory T cell lineage, as opposed to activation-induced transient FOXP3 upregulation. Deep sequencing demonstrated that the TCR repertoir…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported in part by research funding from the Elisabeth Glaser Pediatric AIDS Foundation (Pediatric HIV Vaccine Program Award MV-00-9-900-1429-0-00 to MMA), MGH/ECOR (Physician Scientist Development Award to MMA), NIH NIAID (KO8219 AI074405 and AI074405-03S1 to MMA), and the Harvard University Center for AIDS Research (CFAR), an NIH funded program (P30 AI060354) which is supported by the following NIH Co-Funding and Participating Institutes and Centers: NIAID, NCI, NICHD, NHLBI, NIDA, NIMH, NIA, FIC, and OAR. These studies were furthermore supported by the Bill & Melinda Gates Foundation and the Terry and Susan Ragon Foundation.
Materials
| Reagents | |||
| RosetteSep Human CD4+ T Cell Enrichment Cocktail | Stemcells technologies | 15062 | |
| PBS | Sigma | D8537 | |
| FBS | Sigma | F4135 | |
| Histopaque | Sigma | H8889 | |
| Anti-CD3-PECy7 | BD Pharmingen | 557851 | |
| Anti-CD4-FITC | eBioscience | 11-0049-42 | |
| Anti-CD25-APC | eBioscience | 17-0259-42 | |
| Anti-CD127-PE | BD Pharmingen | 557938 | |
| Round-Bottom tube with 35 μm a nylon mesh | BD Falcon | 352235 | |
| X-VIVO 15 | Lonza | 04-418Q | |
| Penicillin/Streptomycin | Mediatech | 30-001-Cl | |
| Human Serum | Gemini Bio-Products | 100-512 | |
| Human T-activator CD3/CD28 | Life Technologies | 111.31D | |
| IL-2 | NIH Aids Research & Reference Reagent Program | 136 | |
| LIVE/DEAD Fixable Violet Dead Cell Stain Kit | Life technologies | L34955 | |
| Anti-CD4-qdot-655 | Life Technologies | Q10007 | |
| Anti-CD25-PECy5 | eBiosciences | 15-0259-42 | |
| Foxp3 / Transcription Factor Staining Buffer Set | eBiosciences | 00-5523-00 | |
| Anti-FOXP3-PE | eBiosciences | 12-4776-42 | |
| Anti-HELIOS-FITC | Biolegend | 137204 | |
| Anti-CTLA4-APC | BD Pharmingen | 555855 | |
| CellTrace Violet Cell Proliferation Kit | Life Technologies | C34557 | |
| Vybrant CFDA SE Cell Tracer Kit | Life Technologies | V12883 | |
| HEPES | Mediatech | 25-060-Cl | |
| Treg Suppression inspector | Miltenyi Biotec | 130-092-909 | |
| Anti-CD4-APC | BD Pharmingen | 340443 | |
| Anti-CD8-AF700 | BD Pharmingen | 557945 | |
| RPMI 1640 | Sigma | R0883 | |
| Glutamine | Mediatech | 25-002-Cl | |
| Materials | |||
| BD Vacutainer Blood Collection Tube w/ ACID CITRATE DEXTROSE (ACD) | Becton, Dickinson and Company (BD) | 364606 | |
| FACSAria IIu Cell Sorter | BD Biosciences | – | |
| LSR II Flow Cytometer | BD Biosciences | – | |
| FlowJo | Tree Star | v887 |
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