新工具展开调节性T细胞的HIV-1感染的个体

摘要

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.

摘要

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。

引言

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。

研究方案

1. Regulatory T cell isolation from HIV-1 Positive Blood

1. Carefully transfer blood, collected in ACD tubes, into a 50 ml conical tube for a final volume of 15 ml blood per tube.
2. Add 25 μl/ml of blood of RosetteSep Human CD4+ T Cell Enrichment Cocktail, mix carefully and incubate 20 min at room temperature.
3. Add 15 ml of PBS/2% FBS to the blood and mix carefully. Layer the diluted blood sample on top of 15 ml of Histopaque at room temperature in a 50 ml conical tube. Spin the conical tube for 20 min at 1,200 x g with a slow start and no brakes.
4. Transfer the CD4⁺ T cell enriched PBMC layer in a new 50 ml conical tube, wash the cells by adding PBS/2% FBS and spin them down for 10 min at 1,200 x g. Then count the cells, wash again and resuspend the cells at about 20 x 10⁶/200 μl.
5. Add the following antibodies (concentration):
   anti-CD3-Phycoerythrin-Cyanine 7 (PE-Cy7) (1/100)
   anti-CD4-Fluorescein Isothiocyanate (FITC) (1/40)
   anti-CD25-Allophycocyanin (APC) (1/40)
   anti-CD127-Phycoerythrin (PE) (1/20)
   Incubate 30 min in the dark at 4 °C
6. Wash the cells with PBS/2% FBS. Resuspend the cells at 20 x 10⁶/ml in PBS/2% FBS and filter them on a 35 μm nylon mesh.
7. Using a FACS Aria cell sorter equipped for handling biohazardous material, sort the CD3⁺CD4⁺CD25⁺CD127^low Treg in X-VIVO 15 media (see gating strategy in Figure 1). Conventional T cells (CD3⁺CD4⁺CD25^-CD127⁺) can be isolated and expanded as negative controls.

2. Cell Culture

1. After isolation, wash the Treg with X-VIVO 15 media.
2. Resuspend the cells at 250 x 10³/ml in X-VIVO 15 media complemented with 10% Human Serum and Penicillin-Streptomycin (50 U/ml).
3. Wash Human T-Activator CD3/CD28 beads according to manufacturer's protocol. Add beads to isolated Tregs at a ratio of 1:1 bead per cell.
4. After two days of culture, double the media volume and add IL-2 (300 U/ml).
5. Culture the Tregs for 2 weeks. Change media (X-VIVO 15/Human serum/P/S/IL-2) at days 5, 7, 9, 12. Add beads at a 1:1 ratio at day 9. When changing media, keep cells at 250 x 10³/ml.

3. Phenotyping

At the end of the expansion culture, expanded CD3⁺CD4⁺CD25⁺CD127^low Treg can be phenotyped by flow cytometry and compared to expanded CD3⁺CD4⁺CD25^-CD127⁺ conventional T cells as a control.

1. Harvest expanded Tregs/Tconvs and wash them in PBS. Label dead cells using the LIVE/DEAD Fixable Violet Dead Cell Stain Kit according to manufacturer's protocol. Wash the cells in PBS/2% FBS.
2. Add the following antibodies (concentration):
   anti-CD3-PECy7 (1/100)
   anti-CD4-Qdot-655 (1/200)
   anti-CD25-PECy5 (1/100)
   Incubate 30 min in the dark at 4 °C
3. Wash the cells and perform the intracellular staining using the Foxp3/ Transcription Factor Staining Buffer Set according to manufacturer's protocol and the following antibodies:
   anti-FOXP3-PE (1/50)
   anti-HELIOS-FITC (1/40)
   anti-CTLA4-APC (1/20)
   Acquire the data on a flow cytometer.

4. Suppression Assay

At the end of the expansion culture, the suppressive function i.e. the capacity of the expanded Treg isolated from HIV-1 positive individuals to suppress the proliferation of activated T cells can be assessed in vitro.

1. Thaw autologous cryopreserved ex vivo PBMCs. Leave them for about 3 hr in a 37 °C incubator in RPMI 1640 medium containing penicillin/streptomycin, L-glutamine, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (=R+ media), and 10% FBS (=R10 media).
2. Label the dead cells using the LIVE/DEAD Fixable Violet Dead Cell Stain Kit according to the manufacturer's protocol. Wash the cells in PBS/2% FBS.
3. Incubate the cells with anti-CD3-PECy7 for 30 min in the dark at 4 °C. Wash the cells with PBS/2% FBS. Resuspend the cells in PBS/2% FBS and filter them on a 35 μm nylon mesh.
4. Using a FACS Aria cell sorter equipped for handling biohazardous material, sort the viable CD3+ T cells in R10 media.
5. Label the T cells with a cell tracing reagent such as CellTrace Violet or Vybrant CFDA SE Cell Tracer at 5 μM diluted in PBS for 7 min at 37 °C according to the manufacturer's protocol. Resuspend cells in R+ media supplemented with 10% human serum (=hR10 media) at 1 x 10⁶/ml.
6. Harvest the expanded Tregs, resuspend the cells at 0.5 x 10⁶/ml in hR10 and prepare dilutions at 0.25 x 10⁶/ml and 0.125 x 10⁶/ml.
7. Prepare anti-CD2/anti-CD3/anti-CD28 microbeads according to the manufacturer's protocol, resuspend the microbeads at 0.75 x 10⁶/ml and prepare dilutions at 0.625, 0.562, 0.5 x 10⁶/ml in hR10 media.
8. In a 96 wells round bottom plate, transfer cells and beads according to the following plan:

T cells:Treg ratio	1:0	1:1/2	1:1/4	1:1/8
T cells (50 μl)	1 x 106/ml	1 x 106/ml	1 x 106/ml	1 x 106/ml
Tregs (50 μl)	no	0.5 x 106/ml	0.25 x 106/ml	0.125 x 106/ml
Beads (100 μl)	0.5 x 106/ml	0.75 x 106/ml	0.625 x 106/ml	0.562 x 106/ml
hR10 (50 μl)	yes	no	no	no
i.e.
T cells	50 x 103	50 x 103	50 x 103	50 x 103
Tregs	0	25 x 103	12.5 x 103	6.25 x 103
Beads	50 x 103	75 x 103	62.5 x 103	56.25 x 103

9. After 4 days of culture, wash the cells and incubate them for 30 min at 4 °C with the following antibodies:
   anti-CD3-PECy7 (1/100)
   anti-CD4-APC (1/100)
   anti-CD8-AF700 (1/100)

Acquire the data on a flow cytometer. Use the FlowJo proliferation platform to calculate the percentage of divided cells.

结果

The expression of interleukin 2 receptor (CD25) and the interleukin 7 receptor (CD127) have been described as reliable surface markers to identify functional Treg populations ¹³ and have been shown to correlate with CD4⁺CD25⁺FOXP3⁺ Tregs ^9,12. Figure 1 represents the gating strategy used to flow-sort single CD3⁺CD4⁺CD25⁺CD127^low Tregs from PBMC isolated from an HIV-1-positive individual. The CD25/CD127 anti...

讨论

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...

材料

Name	Company	Catalog Number	Comments
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