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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   

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Jia Li
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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.

Keywords: Major histocompatibility complex class II(II类主要组织相容性复合物), 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

  1. Falcon® round-bottom 5 ml polypropylene tubes (Corning, Falcon®, catalog number: 352063 )
  2. Falcon® round-bottom 5 ml polystyrene tubes with a 35 µm Cell-Strainer cap (Corning, Falcon®, catalog number: 352235 )
  3. 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
  4. 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)
  5. Mouse-anti-human CD3 APC-eFluor® 780 (clone UCHT1) (Affymetrix, eBioscience, catalog number: 47-0038-42 )
  6. Mouse-anti-human CD4 BD HorizonTM PE-CF594 (clone RPA-T4) (BD, BD Biosciences, catalog number: 562281 )
  7. Mouse-anti-human CD8 Brilliant Violet 785TM (clone RPA-T8) (BioLegend, catalog number: 301045 )
  8. Mouse-anti-human CD14 VioGreen® (clone TÜK4) (Miltenyi Biotec, catalog number: 130-096-875 )
  9. Mouse-anti-human CD20 VioGreen® (clone LT20) (Miltenyi Biotec, catalog number: 130-096-904 )
  10. Fixable Viability Dye eFluor 506® (Affymetrix, eBioscience, catalog number: 65-0866-14 )
  11. 16% paraformaldehyde (PFA) solution (Electron Microscopy Sciences, catalog number: 15710 )
  12. Phosphate buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  13. RPMI 1604 GlutaMAX ITM (Thermo Fisher Scientific, GibcoTM, catalog number: 61870044 )
  14. HyCloneTM fetal bovine serum (FBS) (South America), research grade (GE Healthcare, catalog number: SV30160.03 )
  15. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  16. 1 M HEPES buffer (Dominique Dutscher SAS, catalog number: P05-01100P )
  17. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3912 )
  18. Sodium azide (NaN3) 5% (w/v) (VWR, BDH®, catalog number: BDH7465-2 )
  19. Human AB serum (PAN-Biotech, catalog number: P30-2901 )
  20. Complete RPMI (see Recipes)
  21. FACS buffer (see Recipes)
  22. Tetramer labeling buffer (see Recipes)
  23. FACS sorting buffer (see Recipes)

Equipment

  1. Benchtop centrifuge (Thermo Fisher Scientific, model: SorvallTM LegendTM XTR )
  2. Benchtop microcentrifuge (Eppendorf, model: 5254 R )
  3. BD LSRFortessaTM cell analyzer (BD, BD Biosciences, model: BD LSRFortessaTM Cell Analyzer )
  4. BD FACSAria IITM flow cytometer (BD, BD Biosciences, model: BD FACSAria IITM Cell Sorter )

Software

  1. FACSDivaTM version 8.0 (BD)
  2. FlowJoTM version 10.2 (FlowJo, LLC)

Procedure

Ex vivo tetramer labeling of CD4+ T cells from HIV infected patients

  1. PBMC from HIV infected samples are either thawed from frozen vials or freshly processed from whole blood in a biosafety level-3 environment.
  2. 15-50 x 106 cells are dispatched into 5 ml polypropylene tubes (5 x 106 cells per tube) and washed once with 2 ml complete RPMI at 600 x g for 7 min at 4 °C.
    Notes:
    1. A wash entails the addition of the indicated volume of the specified washing buffer followed by centrifugation and careful removal of the supernatant.
    2. A minimum of 5 x 106 PBMC is required per tetramer labeling; the same number of PBMC is required for the control tetramer labeling. When scaling up, it is preferable to process multiple identical sample tubes in parallel, so that the amount of cells required for the control tube remains constant at 5 x 106 PBMC.
    3. Individual tubes containing 0.5 x 106 cells in 50 μl FACS buffer should be prepared at this stage to be used as single stained compensation controls. These tubes should be kept at 4 °C for the duration of the tetramer labeling until the point of addition of cell surface antibodies.
  3. Supernatants are removed and the cell pellets are re-suspended in 100 μl pre-chilled tetramer labeling buffer and placed on ice.
  4. Solutions of MHC II tetramers loaded with either a validated HIV-1 peptide, or an irrelevant control peptide (CLIP) are spun at full speed for 5 min at 4 °C to remove protein aggregates.
    Note: Spinning the MHC II tetramer solution before usage reduces background labeling.
  5. APC-conjugated tetramers are added to the cell suspension at 1 μg per 106 cells (i.e., 5 µg per tube).
  6. Important: One tube containing 5 x 106 cells has to be labeled with tetramers loaded with an irrelevant peptide (CLIP) and used as a negative control for tetramer labeling.
  7. Tetramer labeling is performed at 0-4 °C for 60 min, protected from light.
    Note: Staining under cold conditions contributes to reduction of background; however, these conditions are appropriate only for the detection of high-avidity pMHC/TCR interactions.
  8. A mixture of antibodies targeting cell surface markers is added to each tube as follows (v/106 cells): 1 μl CD3 APC-eFluor 780; 0.5 μl CD4 PE-CF594; 0.5 μl CD8 BV 785; 3 μl CD14-VioGreen; 3 μl CD20-VioGreen and 0.5 μl Fixable Viability Dye eFluor 506.
    Notes:
    1. Additional antibodies for cell surface phenotyping can be added at this point.
    2. At this point, tubes containing 0.5 x 106 cells in 50 μl FACS buffer are singly labeled with individual antibodies to be used as compensation controls. For the AmCyan-dump channel (including CD14-VioGreen; CD20-VioGreen and Fixable Viability Dye eFluor 506), a single tube containing 0.5 x 106 cells in 50 μl FACS buffer is labeled with 1.5 μl CD14-VioGreen.
  9. Cells are further incubated with the antibody mixture for 30 min at 4 °C in the dark.
  10. For flow cytometric analysis
    1. Labeled cells are washed twice with (pre-chilled) FACS buffer and fixed with 2% PFA diluted in PBS.
      Note: Fixation entails complete removal of the supernatants following centrifugation and resuspension in 400 µl of the fixation solution.
    2. All cells in the tube are acquired on a flow cytometer (LSR Fortessa).
  11. For cell sorting
    1. Labeled cells are washed once with FACS sorting buffer and the cell concentration is adjusted to 10 x 106 cells per ml in FACS sorting buffer.
    2. Cells are filtered using a Cell-Strainer cap to obtain a single-cell suspension. Subsequently, all cells are acquired on a cell sorter (FACSAria II) installed in a microbiological safety cabinet.
  12. Viable tetramer-labeled CD4+ T cells are visualized in the viable CD3+, CD4+, CD14-, CD20-, CD8- lymphocyte gate (see gating strategy, Figure 1A).
  13. Gates are set according to the negative control, consisting in CLIP-loaded tetramer labeled CD4+ T cells (Figure 1B).


    Figure 1. Ex vivo detection of viable HIV-specific CD4+ T cells from patient PBMC. A. Gating strategy: The Dump channel includes the following antibodies: CD14-VioGreen, CD20-VioGreen and viability dye-eFluor 506. B. FACS plots depicting CD4+ T cells from an HIV-1 infected patient labeled with an HLA DRB5*0101 tetramer loaded either with a control CLIP peptide [PVSKMRMATPLLMQA] (left) or with an immunodominant HIV-1 Gag peptide [FRDYVDRFYKTLRAEQASQE] (right).

Data analysis

Flow cytometry data was analyzed with the Flowjo v10.2 software (FlowJo, LLC.).

Recipes

  1. Complete RPMI
    440 ml RPMI 1640
    50 ml FBS
    5 ml penicillin-streptomycin
    5 ml HEPES
  2. FACS buffer (For 1 L)
    982 ml 1x PBS
    10 g BSA
    18 ml 5% (w/v) sodium azide
  3. Tetramer labeling buffer (1 ml)
    850 μl RPMI (no FBS)
    150 μl human AB serum
  4. FACS sorting buffer (50 ml)
    49.5 ml 1x PBS
    0.5 ml FBS

Acknowledgments

This protocol was adapted from Benati et al. (2016). We acknowledge funding from Agence Nationale de la Recherche sur le SIDA et les Hépatites Virales (ANRS EP36-8) and Agence Nationale de la Recherche (ANR PD1VAX).

References

  1. Benati, D., Galperin, M., Lambotte, O., Gras, S., Lim, A., Mukhopadhyay, M., Nouel, A., Campbell, K. A., Lemercier, B., Claireaux, M., Hendou, S., Lechat, P., de Truchis, P., Boufassa, F., Rossjohn, J., Delfraissy, J. F., Arenzana-Seisdedos, F. and Chakrabarti, L. A. (2016). Public T cell receptors confer high-avidity CD4 responses to HIV controllers. J Clin Invest 126(6): 2093-2108.
  2. Seth, N., Kaufmann, D., Lahey, T., Rosenberg, E. S. and Wucherpfennig K. W. (2005). Expansion and contraction of HIV-specific CD4 T cells with short bursts of viremia, but physical loss of the majority of these cells with sustained viral replication. J Immunol 175(10): 6948-58.

简介

主要组织相容性复合物(MHC)四聚体已经使用二十年来检测,分离和表征各种病原体和肿瘤抗原特异性的T细胞。在人类免疫缺陷病毒(HIV)感染的背景下,抗原特异性CD8 +细胞已经在体外广泛研究,因为它们可以容易地被HIV肽 - 加载MHC I类四聚体。相比之下,HIV特异性CD4 + sup + T细胞的检测已被证明更具挑战性,因为CD4 + sup + T细胞的本质上较低的克隆扩增率以及优先艾滋病毒感染过程中艾滋病毒特异性CD4 + T细胞的消耗。
 在以下协议中,我们描述了一种简单的方法,该方法有助于使用肽负载的MHC II类四聚体来鉴定HIV-1衣壳表位特异性的CD4 + / T细胞。可以分析四聚体标记的CD4
T细胞的细胞表面表型和/或FACS分选用于进一步的下游应用。成功检测特异性CD4 + / / T细胞离体的关键是选择导致高亲和力T细胞受体(TCR)的肽/ MHC II组合, (Benati等人,2016)。 MHC II四聚体阳性细胞的可靠检测的第二个关键点是系统地使用负载无关肽的对照四聚体,样品和对照管在相同的条件下进行处理。

背景 在用抗PE微珠对四聚体-PE标记的细胞进行磁力富集后,在纯化的CD4 + T细胞中检测到罕见的HIV特异性MHC II四聚体阳性细胞(Seth等人, em>。,2005)。我们发现使用经验证的肽/ MHC II四聚体组合,一种简单的基于5×10 6个患者外周血单核细胞(PBMC)直接四聚体标记的方案,随后在流程上获得所有事件细胞计数仪可以很好地检测HIV特异性CD4 + T细胞。通过适当的门控策略排除不相关细胞(CD14 + ,CD20 + ,CD8 + )和死细胞(可固定活性染色剂+)改进标签特异性。

关键字:II类主要组织相容性复合物, 四聚体, T细胞受体, CD4+ T细胞, HIV

材料和试剂

  1. Falcon ®圆底5ml聚丙烯管(Corning,Falcon ®,目录号:352063)
  2. Falcon ®圆底5毫升具有35微米细胞过滤器帽的恒定管(Corning,Falcon ®,目录号:352235)
  3. APC-缀合的抗原负载的MHC II四聚体可以通过NIH Tetramer Core Facility,Emory University,USA获得。提供浓度为1-1.5mg/ml范围的四聚体,等份为200μl
  4. 装载有不相关肽(通常CLIP肽:PVSKMRMATPLLMQA可以通过NIH Tetramer核心设施,Emory University,USA获得)的APC-缀合的MHC II四聚体
  5. 小鼠抗人CD3 APC-eFluor(克隆UCHT1)(Affymetrix,eBioscience,目录号:47-0038-42)
  6. 小鼠抗人CD4 BD Horizon PE-CF594(克隆RPA-T4)(BD,BD Biosciences,目录号:562281)
  7. (克隆RPA-T8)(BioLegend,目录号:301045)
    小鼠抗人CD8亮蓝785 TM
  8. 小鼠抗人CD14 VioGreen ®(克隆TÜK4)(Miltenyi Biotec,目录号:130-096-875)
  9. 小鼠抗人CD20 VioGreen (克隆LT20)(Miltenyi Biotec,目录号:130-096-904)
  10. 可固定的活性染料eFluor 506 ®(Affymetrix,eBioscience,目录号:65-0866-14)
  11. 16%多聚甲醛(PFA)溶液(Electron Microscopy Sciences,目录号:15710)
  12. 磷酸盐缓冲盐水(PBS),pH 7.4(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
  13. RPMI 1604 GlutaMAX I TM(Thermo Fisher Scientific,Gibco TM,目录号:61870044)
  14. HyClone TM胎牛血清(FBS)(南美),研究级(GE Healthcare,目录号:SV30160.03)
  15. 青霉素 - 链霉素(10,000U/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  16. 1 M HEPES缓冲液(Dominique Dutscher SAS,目录号:P05-01100P)
  17. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A3912)
  18. 叠氮化钠(NaN3)5%(w/v)(VWR,BDH ,目录号:BDH7465-2)
  19. 人AB血清(PAN-Biotech,目录号:P30-2901)
  20. 完成RPMI(见配方)
  21. FACS缓冲区(见配方)
  22. 四聚体标记缓冲液(见配方)
  23. FACS排序缓冲区(见配方)

设备

  1. 台式离心机(Thermo Fisher Scientific,型号:Sorvall TM Legend TM XTR)
  2. 台式微量离心机(Eppendorf,型号:5254 R)
  3. BD LSRFortessa TM细胞分析仪(BD,BD Biosciences,型号:BD LSRFortessa TM细胞分析仪)
  4. 流式细胞仪(BD,BD Biosciences,型号:BD FACSAria II 细胞分选机)的BD FACSAria II TM

软件

  1. FACSDiva TM 版本8.0(BD)
  2. FlowJo TM 版本10.2(FlowJo,LLC)

程序

HIV感染患者CD4 + sup + T细胞的体外四聚体标记

  1. 来自HIV感染样本的PBMC或者从冷冻小瓶中解冻,或者在生物安全3级环境中全血新鲜处理。
  2. 将15-50×10 6个细胞分配到5ml聚丙烯管(每管5×10 6个细胞)中,并用2ml完全RPMI在600℃洗涤一次,在4℃下,xg 7分钟。
    注意:
    1. 洗涤需要加入指定体积的指定的洗涤缓冲液,然后离心并仔细去除上清液。
    2. 每个四聚体标记需要至少5×10 6个/个PBMC;控制四聚体标签需要相同数量的PBMC。当放大时,优选并行处理多个相同的样品管,使得对照管所需的细胞数量保持恒定在5×10 6个PBMC。 >
    3. 在50μlFACS缓冲液中含有0.5×10 6个细胞的单个管应在此阶段制备,用作单染色补偿对照。这些管应保持在4℃持续四聚体标记的持续时间,直到细胞表面抗体加入点。
  3. 去除上清液,将细胞沉淀重新悬浮在100μl预冷的四聚体标记缓冲液中并置于冰上。
  4. 装载有验证的HIV-1肽或不相关对照肽(CLIP)的MHC II四聚体的溶液在4℃以全速旋转5分钟以除去蛋白质聚集体。
    注意:在使用MHC II四聚体溶液之前,请先转载背景标签。
  5. 将APC-缀合的四聚体以每10 6个细胞(即,每管5μg)1μg加入细胞悬液中。
  6. 重要信息:含有5×10 6个细胞的一条细胞必须用装有不相关肽(CLIP)的四聚体进行标记,并用作四聚体标记的阴性对照。
  7. 四聚体标记在0-4℃下进行60分钟,防止光照。
    注意:在寒冷条件下染色有助于减少背景;然而,这些条件仅适用于检测高亲合力pMHC/TCR相互作用。
  8. 将如下针对细胞表面标志物的抗体混合物加入到每个管中(v/10 6个细胞):1μlCD3 APC-eFluor 780; 0.5μlCD4 PE-CF594; 0.5μlCD8 BV 785; 3μlCD14-VioGreen; 3μlCD20-VioGreen和0.5μl可固定活性染料eFluor 506。
    注意:
    1. 此时可以添加细胞表面表型的其他抗体。
    2. 此时,在50μlFACS缓冲液中含有0.5×10 6个细胞的管子用单独的抗体单独标记以用作补偿对照。对于AmCyan-dump通道(包括CD14-VioGreen; CD20-VioGreen和Fixable Viability Dye eFluor 506),在50μlFACS缓冲液中含有0.5×10 6个细胞的单管用1.5μlCD14 -VioGreen。
  9. 将细胞与抗体混合物在4℃下在黑暗中进一步温育30分钟
  10. 流式细胞仪分析
    1. 标记的细胞用(预冷)FACS缓冲液洗涤两次,并用在PBS中稀释的2%PFA固定。
      注意:固定需要在离心后完全去除上清液,并在400μl固定液中重新悬浮。
    2. 管中的所有细胞在流式细胞仪(LSR Fortessa)上获得。
  11. 用于细胞分选
    1. 标记的细胞用FACS分选缓冲液洗涤一次,并将细胞浓度在FACS分选缓冲液中调节至每毫升10×10 6个细胞。
    2. 使用细胞 - 过滤器帽过滤细胞以获得单细胞悬浮液。随后,将所有细胞在安装在微生物安全柜中的细胞分选仪(FACSAria II)上获得。
  12. 可行的四聚体标记的CD4 + T细胞在可行的CD3 +,> CD4 +,CD14 -/- CD20 - ,CD8 - 淋巴细胞门(参见门控策略,图1A)。
  13. 根据阴性对照设置门,其中包含CLIP-负载四聚体标记的CD4 + T细胞(图1B)。


    图1.来自患者PBMC的活的HIV特异性CD4 + T细胞的体外检测。 A.门控策略:转储通道包括以下抗体:CD14-VioGreen,CD20-VioGreen和存活性染料-EFluor506.BFACS图描绘来自HIV-1感染患者的CD4 + sup + T细胞,其用HLA DRB5 * 0101四聚体标记, (左侧)或具有免疫显性HIV-1 Gag肽[FRDYVDRFYKTLRAEQASQE](右)的对照CLIP肽[PVSKMRMATPLLMQA](左)。

数据分析

流式细胞术数据用Flowjo v10.2软件(FlowJo,LLC。)分析。

食谱

  1. 完成RPMI
    440 ml RPMI 1640
    50ml FBS
    5 ml青霉素 - 链霉素
    5 ml HEPES
  2. FACS缓冲区(1 L)
    982 ml 1x PBS
    10克BSA
    18毫升5%(w/v)叠氮化钠
  3. 四聚体标记缓冲液(1 ml)
    850μlRPMI(无FBS)
    150μl人类AB血清
  4. FACS分选缓冲液(50ml)
    49.5ml 1x PBS
    0.5 ml FBS

致谢

该协议由Benati等人改编。 (2016年)。我们承认来自国际老年痴呆症患者(ANRS EP36-8)和国民健康计划(ANR PD1VAX)的资助。

参考文献

  1. Benati,D.,Galperin,M.,Lambotte,O.,Gras,S.,Lim,A.,Mukhopadhyay,M.,Nouel,A.,Campbell,KA,Lemercier,B.,Claireaux,M.,Hendou ,S.,Lechat,P.,de Truchis,P.,Boufassa,F.,Rossjohn,J.,Delfraissy,JF,Arenzana-Seisdedos,F.and Chakrabarti,LA(2016)。< a class = ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27111229"target ="_ blank">公共T细胞受体赋予HIV控制器高亲和力CD4反应。 em> J Clin Invest 126(6):2093-2108。
  2. Seth,N.,Kaufmann,D.,Lahey,T.,Rosenberg,ES和Wucherpfennig KW(2005)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih .VV/pubmed/16272355"target ="_ blank">病毒血症短暂的艾滋病毒特异性CD4T细胞的扩张和收缩,但具有持续病毒复制的大多数这些细胞的物理损失。 J Immunol 175(10):6948-58。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
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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