MHC Class II Tetramer Labeling of Human Primary CD4+ T Cells from HIV Infected Patients
使用MHC II类四聚体标记来自HIV感染患者的人原代CD4+ T细胞
发布: 2017年03月20日第7卷第6期 DOI: 10.21769/BioProtoc.2187 浏览次数: 10632
评审: Jia LiGuangzhi ZhangAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jun 2016
Abstract
Major Histocompatibility Complex (MHC) tetramers have been used for two decades to detect, isolate and characterize T cells specific for various pathogens and tumor antigens. In the context of Human Immunodeficiency Virus (HIV) infection, antigen-specific CD8+ T cells have been extensively studied ex vivo, as they can be readily detected by HIV peptide-loaded MHC class I tetramers. In contrast, the detection of HIV-specific CD4+ T cells has proven more challenging, due to the intrinsically lower clonal expansion rates of CD4+ T cells, and to the preferential depletion of HIV-specific CD4+ T cells in the course of HIV infection.
In the following protocol, we describe a simple method that facilitates the identification of CD4+ T cells specific for an HIV-1 capsid epitope using peptide-loaded MHC class II tetramers. Tetramer labeled CD4+ T cells can be analyzed for their cell surface phenotype and/or FACS-sorted for further downstream applications. A key point for successful detection of specific CD4+ T cells ex vivo is the choice of a peptide/MHC II combination that results in high-affinity T Cell Receptor (TCR) binding (Benati et al., 2016). A second key point for reliable detection of MHC II tetramer-positive cells is the systematic use of a control tetramer loaded with an irrelevant peptide, with the sample and control tubes being processed in identical conditions.
Tetramer (四聚体)
T cell receptor (T细胞受体)
CD4+ T cell (CD4+ T细胞)
HIV (HIV)
Background
Rare HIV-specific MHC II tetramer-positive cells have been detected in purified CD4+ T cells, after magnetic enrichment of tetramer-PE labeled cells with anti-PE microbeads (Seth et al., 2005). We found that with validated peptide/MHC II tetramer combinations, a simpler protocol based on direct tetramer labeling of 5 x 106 patient Peripheral Blood Mononuclear Cells (PBMC), followed by acquisition of all events on a flow cytometer, resulted in reliable detection of HIV-specific CD4+ T cells. Exclusion of irrelevant cells (CD14+, CD20+, CD8+) and dead cells (Fixable Viability dye+) through an appropriate gating strategy improved labeling specificity.
Materials and Reagents
- Falcon® round-bottom 5 ml polypropylene tubes (Corning, Falcon®, catalog number: 352063 )
- Falcon® round-bottom 5 ml polystyrene tubes with a 35 µm Cell-Strainer cap (Corning, Falcon®, catalog number: 352235 )
- APC-conjugated antigen-loaded MHC II tetramers can be obtained through the NIH Tetramer Core Facility, Emory University, USA. The tetramers are provided at a concentration in the 1-1.5 mg/ml range, in aliquots of 200 µl
- APC-conjugated MHC II tetramers loaded with an irrelevant peptide (usually the CLIP peptide: PVSKMRMATPLLMQA can be obtained through the NIH Tetramer Core Facility, Emory University, USA)
- Mouse-anti-human CD3 APC-eFluor® 780 (clone UCHT1) (Affymetrix, eBioscience, catalog number: 47-0038-42 )
- Mouse-anti-human CD4 BD HorizonTM PE-CF594 (clone RPA-T4) (BD, BD Biosciences, catalog number: 562281 )
- Mouse-anti-human CD8 Brilliant Violet 785TM (clone RPA-T8) (BioLegend, catalog number: 301045 )
- Mouse-anti-human CD14 VioGreen® (clone TÜK4) (Miltenyi Biotec, catalog number: 130-096-875 )
- Mouse-anti-human CD20 VioGreen® (clone LT20) (Miltenyi Biotec, catalog number: 130-096-904 )
- Fixable Viability Dye eFluor 506® (Affymetrix, eBioscience, catalog number: 65-0866-14 )
- 16% paraformaldehyde (PFA) solution (Electron Microscopy Sciences, catalog number: 15710 )
- Phosphate buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
- RPMI 1604 GlutaMAX ITM (Thermo Fisher Scientific, GibcoTM, catalog number: 61870044 )
- HyCloneTM fetal bovine serum (FBS) (South America), research grade (GE Healthcare, catalog number: SV30160.03 )
- Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
- 1 M HEPES buffer (Dominique Dutscher SAS, catalog number: P05-01100P )
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3912 )
- Sodium azide (NaN3) 5% (w/v) (VWR, BDH®, catalog number: BDH7465-2 )
- Human AB serum (PAN-Biotech, catalog number: P30-2901 )
- Complete RPMI (see Recipes)
- FACS buffer (see Recipes)
- Tetramer labeling buffer (see Recipes)
- FACS sorting buffer (see Recipes)
Equipment
- Benchtop centrifuge (Thermo Fisher Scientific, model: SorvallTM LegendTM XTR )
- Benchtop microcentrifuge (Eppendorf, model: 5254 R )
- BD LSRFortessaTM cell analyzer (BD, BD Biosciences, model: BD LSRFortessaTM Cell Analyzer )
- BD FACSAria IITM flow cytometer (BD, BD Biosciences, model: BD FACSAria IITM Cell Sorter )
Software
- FACSDivaTM version 8.0 (BD)
- FlowJoTM version 10.2 (FlowJo, LLC)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Galperin, M., Benati, D., Claireaux, M., Mukhopadhyay, M. and Chakrabarti, L. A. (2017). MHC Class II Tetramer Labeling of Human Primary CD4+ T Cells from HIV Infected Patients. Bio-protocol 7(6): e2187. DOI: 10.21769/BioProtoc.2187.
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分类
免疫学 > 免疫细胞染色 > 流式细胞术
细胞生物学 > 细胞染色 > 其它化合物
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