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Analysis of Tumor-infiltrating Lymphocytes Following CD45 Enrichment
CD45富集后的肿瘤浸润淋巴细胞的分析   

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Abstract

Measuring antigen-specific T cell responses in the blood and lymphoid organs of vaccinated mice can give us a useful indication of the potency of a vaccine formulation. Unfortunately, systemic or even localized lymphoid T cell responses are not always predictive of the ability of a vaccine to induce tumor protection. Measuring the antigen-specific T cell response within the tumor infiltrating lymphocytes is a more accurate indicator or vaccine efficacy. However, multi-parameter flow cytometric analysis of T cells isolated from tumor tissue can be quite challenging due to the over-whelming number of tumor cells present in relation to the tumor infiltrating lymphocytes (TIL) and to problems associated to the large and adhesive nature of many tumor cell. Here we take advantage of a pre-flow separation of CD45+ leukocytes from the tumor tissue using the MACS magnetic cell sorting system, resulting in a much cleaner cell preparation with which to proceed to flow cytometric staining and analysis.

Keywords: TIL(直到), Tumor-infiltrating lymphocytes(肿瘤浸润淋巴细胞), B16 melanoma(B16黑色素瘤), CD45 enrichment(CD45富集), CD8+ T cells(CD8 + T细胞)

Materials and Reagents

  1. Cell lines
    1. The B16.OVA melanoma cell line (Fidler, 1975; Schuler et al., 2008) maintained in cDMEM supplemented with G418-sulphate (geneticin selective antibiotic) (at 1 mg/ml)
      Note: Adherent cells detached using 0.25 % Trypsin-EDTA.

  2. Mice
    1. All mice used in this protocol were between 6 and 12 weeks of age and were sex and age matched for each individual experiment. Three mice were used per group in each experiment.
    2. CD45.1 congenic (B6.SJL-PtprcaPep3b/BoyJArc), bred in-house at the SPF Animal Facility of the UNIL
    3. OT-I mice (Hogquist et al., 1994)
    4. OT-IIxFoxp3-eGFP mice [referred to as OT-II (Barnden et al., 1998, Wang et al., 2008)] bred in-house at the SPF Animal Facility of the UNIL

  3. Antibodies
    1. Va2 and Vb5.1/5.2 antibodies (BD Biosciences)
    2. CD45 PE antibody (BD Biosciences)
    3. CD45.1 APC-eFluor780 (eBioscience)
    4. CD45.2 pacific blue (eBioscience)
    5. CD8 eFluor700 (eBioscience)
    6. CD4 PE-Texas red (eBioscience)
    7. 2.4G2 (Anti-FcgRII monoclonal antibody)

  4. Buffers and media
    1. Dulbecco’s modified Eagle’s medium (DMEM), high glucose, GlutaMAX™ supplement (Life Technologies, Gibco®, catalog number: 10566-016 )
    2. 1 M HEPES (Life Technologies, Gibco®, catalog number: 15630-080 )
    3. Penicillin-streptomycin (5,000 U/ml) (Life Technologies, Gibco®, catalog number: 15070063 )
    4. Fœtal bovine serum (FBS). (performance sera with low endotoxin : qualified, US origin) (Life Technologies, catalog number: 26140 or similar)
    5. Geneticin® selective antibiotic (G418 Sulfate, 50 mg/ml) (Life Technologies, Gibco®, catalog number: 15630-080)
    6. 0.25% Trypsin-EDTA  (1x) (phenol red) (Life Technologies, InvitrogenTM, catalog number: 25200056 )
    7. Phosphate buffered saline (PBS) (Laboratorium Dr. Bichsel AG)
    8. Collagenase, Type I (Life Technologies, Gibco®, catalog number: 17018-029 )
    9. DNAse I  (Roche Diagnostics, catalog numner: 0 4536282001 )
    10. UltraPure™ 0.5 M EDTA (pH 8.0) (Life Technologies, InvitrogenTM, catalog number: 15575-020 )
    11. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
    12. Complete Dulbecco’s modified Eagle’s medium (cDMEM) (see Recipes)
    13. Tumor digesting buffer (see Recipes)
    14. Flow Buffer (see Recipes)
    15. MACS Buffer (see Recipes)

  5. Peptides
    1. OVA257-264 and OVA323-339 peptides
      Note: Peptides were manufactured by the Protein and Peptide Chemistry Facility (PPCF) of the UNIL.

  6. Adjuvants
    1. Poly (I: C) HMW  (tlrl-pic) and  Imiquimod R837 (tlrl-imq, InvivoGen)
    2. CpG-ODN 1826 (Coley Pharmaceuticals. No longer available. CpG-ODN 1826, tlrl-1826 from InvivoGen can be substituted.)
    3. Quil A saponin mix from Quillaja saponaria (Brenntag Nordic A/S)

  7. Commercial reagents
    1. CD45 MACS microbeads, mouse (Miltenyi Biotec, catalog number: 130-052-301 )
      Datasheet available online: https://www.miltenyibiotec.com/~/media/Images/Products/Import/0001200/IM0001245.ashx
    2. LIVE/DEAD Aqua cell stain (Life Technologies, InvitrogenTM, catalog number: L34957 )

Equipment

  1. Falcon™ 40 µm cell strainer (blue) (Corning, catalog number: 352340 )
  2. 1ml BD Tuberculin Syringe & 26 g or 27 g x 0.5" BD™ PrecisionGlide Needle (BD, catalog number: 3052111 or 305109 )
  3. Falcon™ 50 ml conical centrifuge tubes (Corning, catalog number: 352070 )
  4. Falcon™ 15 ml conical centrifuge tubes (Corning, catalog number: 352099 )
  5. Falcon™ 6 well, non-treated, flat-bottom tissue culture (Corning, catalog number: 351147 )
  6. Dissection scissors or scalpel blade and fine-tipped forceps or tweezers
  7. 10 ml BD™ syringe (catalog number: 309604 ) with 18 g or 20 g x 1" BD™ PrecisionGlide needle (BD, catalog number: 305195 or 305175 )
  8. Haemocytometer
  9. AutoMACS automatic cell separator (Miltenyi Biotech)
  10. FACSCanto flow cytometers (BD)

Software

  1. FlowJo software for Mac, version 9 software (TreeStar)
  2. Prism Graphpad software
    Note: It was used to perform One-way Anova test combined with the Dunnet’s post test for statistical analysis of 3 samples per group, per experiment. Experiments were repeated twice for statistical robustness.

Procedure

  1. Adoptive cell transfer, tumor challenge and immunization
    1. Adoptive cell transfers
      Antigen-specific CD8+ (OT-I) and CD4+ (OT-II) T cells were isolated from spleens of CD45.2+ T cell receptor (TCR)-transgenic mice. Spleens were disrupted over 40 µm cell strainers with the plastic end of a 1 ml tuberculin syringe plunger and collected into 50 ml Falcon tubes. Total cell numbers were determined by counting with a Haemocytometer. The frequency of OT-I and OT-II cells in the spleens was determined by labelling with TCR Va2 and Vb5.1/5.2 antibodies and analysis by flow cytometry before injection of total splenocytes containing the desired number of antigen-specific T cells (Figure 1). Naive CD45.1 recipient mice received 1e6 OT-I cells and 3e6 OT-II cells in 200 µl of DMEM intravenously in the caudal vein.
      Note: T cells can also be transferred by intra-orbital or intra-peritoneal injection if preferred.
    2. Tumor challenge
      Mice were challenged the next day with 2e5 B16.OVA tumor cells in 100 µl PBS injected subcutaneously in the left flank. Briefly, mice were restrained using a single-handed hold. About 2/3 of the length of the needle of the tuberculin syringe was inserted into the subcutaneous layer of skin proximal to the joint of the hind leg, at an upward and sideways angle. The correct position of the end of the needle was checked by gently moving the bevel below the skin (just about the hip joint) and confirming it was in loose space. The tumor cells were slowly injected in a single location. The needle was withdrawn carefully and the exit point massaged slightly with the fingertip to prevent escape of the tumor cells.
    3. Immunisations
      1. One week later, once tumors were palpable, mice were immunised with 10 µg OVA257-264 and 10 µg OVA323-339 peptides in 100 µl PBS subcutaneously at the base of the tail with a 1 ml tuberculin syringe. Briefly, mice were restrained in a holding tunnel with an opening at the end to access the base of the tail. About half the length of the needle of the tuberculin syringe was inserted into the subcutaneous layer of skin just above and to the side of the tail. The correct depth was confirmed by visualizing the bevel just below the skin. The vaccine was slowly injected while keeping pressure at the needle entry point.
        Note: A bubble of liquid should become visible under the skin if the correct technique is applied.
      2. Peptides were injected alone or in combination with 50 µg of one of the following adjuvants: CpG-ODN (CpG), HMW Poly (I: C), imiquimod, or Quil A.

  2. Tissue harvest and processing
    1. One week after vaccination, mice were euthanized by CO2 asphyxiation followed by cervical dislocation. Tumors were exposed by cutting through the skin of the flank and excised by carefully grasping with fine-tipped forceps and snipping away the surrounding skin and connective tissue using sharp dissection scissors.  Tumors were then placed in pre-weighed 15 ml Falcon tubes containing 2 ml PBS.
    2. Tubes containing tumors were weighed again and the initial weight of the tube and buffer subtracted to determine tumor mass.
    3. Tumors larger than 500 µg were cut down to ~500 µg for processing, to avoid overloading of MACS column and as the number of cells obtained in larger tumors are in excess of requirement for flow cytometric analysis.
    4. Tumors were cleaned of connective tissue and each tumor was placed in a 6 well plate in 1 ml of fresh Tumor digesting buffer. Tumors were dissected by chopping with scissors or by slicing repeatedly with a scalpel blade to produce pieces of ~2 mm2 or less. A further 9 ml of Tumor digesting buffer was added to each well and mixed with the tissue by pipetting gently.
    5. Tumor tissue was then incubated at 37 °C for one hour to allow digestion, swirling the plate or pipetting the suspension up and down after 30 min to mix.
    6. At the end of the incubation the tissue pieces were aspirated several times through a 10 ml syringe with a large bore needle (18-20 gauge) to break up remaining tissue and obtain a single cell suspension.
    7. Cells were washed 2-3 times in 50 ml cDMEM and centrifuged at 1,500 rpm for 10 min to remove excess melanin.
    8. A rough total cell count was performed using a haemocytometer to determine the amount of MACS beads required.

  3. CD45+ cell selection by magnetic cell sorting with the AutoMACS
    1. Tumor cells were transferred to 50 ml Falcon tubes and resuspended in 90 µl MACS buffer per 107 cells, as per the manufacturer’s protocol.
    2. 10 µl anti-CD45 beads were added per 107 cells and the cell and bead mixture was incubated on ice for 15 min with mixing by inversion every 5 min.
    3. Cells were then washed twice in 50 ml MACS buffer to remove excess beads.
    4. Cells were then resuspended in 500 µl MACS buffer by gentle pipetting and passed over a 40 µm cell strainer to remove any remaining clumps.
    5. CD45+ cells were purified by positive selection using magnetic cell separation (MACS) beads and the AutoMACS automatic cell separator. Cell supensions were mixed by vortex at low speed just before passing them through the autoMACS system using a POSSEL program.
    6. The positive fraction was counted using a haemocytometer to determine the total TIL number before proceeding to FACS staining of the cells.
      Notes:
      1. The POSSEL program passes the sample through the autoMACS using a single pass over one magnetic column and is designed to obtain a particular cell population by positive selection from a sample in which a normal to high frequency of the cells express the antigen of interest.
      2. If an autoMACS machine is not available then manual MACS columns (Miltenyi Biotech) may be used instead. The manual separation procedure can be found in the CD45 MACS microbead datasheet (see section G).
      3. The purity and yield of the sort can be checked by labeling a small aliquot of the pre-sort and post-sort positive and negative fractions with a CD45 PE antibody and analysis by flow cytometry (Figure 2). After the first 2 experiments, yielding >90% CD45+ cells in the positive fraction, purity testing was no longer performed before the global FACS analysis.

  4. Flow cytometry
    1. ~5 x 106 cells (or the whole sample if less than this number were obtained) were transferred to a 96 well u-bottom plate and washed with PBS.
    2. Cells were resuspended in 100 µl Live/dead Aqua cell stain at a 1/250 dilution in PBS and incubated for 20 min before washing twice with PBS.
    3. 25 µl of supernatant from a 2.4G2 (anti-FcgRII monoclonal antibody) producing hybridoma (grown in-house) was added to the samples and incubated on ice for 5-10 min to inhibit nonspecific antibody binding.
    4. A mix of fluorescent antibodies at 2x final concentration was made in Flow buffer and 25 µl of the mix was added to the cells without first removing the 2.4G2.
    5. Cells were incubated for a further 20-30 min and the plate was then washed twice with 200 µl per well of Flow buffer.
    6. Cells were resuspended in 200 µl of Flow buffer and kept on ice until analysis.
    7. Samples were acquired using LSR-II and FACSCanto flow cytometers.
    8. Lymphocytes were gated on the basis of forward scatter and side scatter properties and LIVE/DEAD Aqua cell stain was used to exclude dead cells.
    9. Flow data was analyzed using FlowJo software (Figure 3).

  5. Multi-colour staining panel
    1. Live/dead dye Aqua cell stain.
    2. CD45.1 APC-eFluor780, CD45.2 Pacific Blue, CD8 eFluor700, CD4 PE-Texas Red (optional labeling with Va2 APC and Vb5.1/5.2 PE for more precise identification of TCR transgenic T cells).
    3. Foxp3 eGFP (endogenous expression).
    4. As well as the multi-colour staining mix used to label the samples, single stains were made for each fluorochrome (and blood from a Foxp3-eGPF mouse was extracted by nicking of the caudal vein for the GFP single stain).
    5. The auto compensation option in the BD Diva software was used to compensate the spectral overlap between all the different fluorochromes.

Representative data



Figure 1. Determining TCR transgenic T cell frequency by Flow cytometry prior to adoptive T cell transfer (lymphocyte population gated based on forward and side scatter properties)


Figure 2. CD45 cell purity after MACS positive selection (single cells first gated based on forward scatter height vs area and live cells detected using Live/dead dye Aqua cell stain)


Figure 3. Representative Flow plots showing progressive gating strategy for analysis of tumor infiltrating lymphocytes in CD45 MACS-enriched tumor samples

Recipes

  1. Complete medium (cDMEM)
    DMEM
    5% FBS
    100 U/ml penicillin & 100 μg/ml streptomycin
    25mM Hepes
    55 μM 2-ME
  2. Tumor digesting buffer
    DMEM
    4.4 mg/ml Collagenase I
    10 µg /ml DNase I
  3. Flow buffer
    PBS
    2% FBS
  4. MACS buffer
    PBS
    1% BSA
    10 mM EDTA

Acknowledgments

Abbreviated protocol previously published in: Perret et al. (2013). This work was supported by grants from the New Zealand Foundation for Research Science and Technology and the Emma Muschamp Foundation (R. Perret) and from the Swiss National Science Foundation (310030-130812 and CRSII3_141879) and the Medic Foundation (P. Romero). Disclosure of Potential Conflicts of Interest: 
P. Romero is a consultant/advisory board member of Immatics Biotechnologies, DC Prime, Matwin, and Center for Human Immunology, Pasteur Institute (Paris, France). No potential conflicts of interest were disclosed by the other authors.

References

  1. Barnden, M. J., Allison, J., Heath, W. R. and Carbone, F. R. (1998). Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements. Immunol Cell Biol 76(1): 34-40.
  2. Fidler, I. J. (1975). Biological behavior of malignant melanoma cells correlated to their survival in vivo. Cancer Res 35(1): 218-224.
  3. Hogquist, K. A., Jameson, S. C., Heath, W. R., Howard, J. L., Bevan, M. J. and Carbone, F. R. (1994). T cell receptor antagonist peptides induce positive selection. Cell 76(1): 17-27.
  4. Perret, R., Sierro, S. R., Botelho, N. K., Corgnac, S., Donda, A. and Romero, P. (2013). Adjuvants that improve the ratio of antigen-specific effector to regulatory T cells enhance tumor immunity. Cancer Res 73(22): 6597-6608.
  5. Schuler, P., Contassot, E., Irla, M., Hugues, S., Preynat-Seauve, O., Beermann, F., Donda, A., French, L. E. and Huard, B. (2008). Direct presentation of a melanocyte-associated antigen in peripheral lymph nodes induces cytotoxic CD8+ T cells. Cancer Res 68(20): 8410-8418.
  6. Wang, Y., Kissenpfennig, A., Mingueneau, M., Richelme, S., Perrin, P., Chevrier, S., Genton, C., Lucas, B., DiSanto, J. P., Acha-Orbea, H., Malissen, B. and Malissen, M. (2008). Th2 lymphoproliferative disorder of LatY136F mutant mice unfolds independently of TCR-MHC engagement and is insensitive to the action of Foxp3+ regulatory T cells. J Immunol 180(3): 1565-1575.

简介

测量接种疫苗的小鼠的血液和淋巴器官中的抗原特异性T细胞应答可以给予我们疫苗制剂的效力的有用指示。不幸的是,全身性或甚至局部的淋巴T细胞应答并不总是预示疫苗诱导肿瘤保护的能力。测量肿瘤浸润淋巴细胞内的抗原特异性T细胞应答是更准确的指示或疫苗功效。然而,从肿瘤组织分离的T细胞的多参数流式细胞术分析可能是相当具有挑战性的,因为相对于肿瘤浸润性淋巴细胞(TIL)存在的肿瘤细胞的数量过多,以及与大的和粘附性质相关的问题的许多肿瘤细胞。在这里我们利用使用MACS磁性细胞分选系统从肿瘤组织中预流动分离CD45 +白细胞,产生更清洁的细胞制备物,进行流式细胞染色和分析。

关键字:直到, 肿瘤浸润淋巴细胞, B16黑色素瘤, CD45富集, CD8 + T细胞

材料和试剂

  1. 细胞系
    1. B16.OVA黑素瘤细胞系(Fidler,1975; Schuler等人,2008)保持在补充有G418-硫酸盐(遗传霉素选择性抗生素)(1mg/ml)的cDMEM中
      注意:使用0.25%胰蛋白酶-EDTA分离粘附细胞。

  2. 老鼠
    1. 在该方案中使用的所有小鼠在6-12周龄之间,并且对于每个个体实验是性别和年龄匹配的。 在每个实验中每组使用三只小鼠
    2. CD45.1同源物(B6.SJL-PtprcaPep3b/BoyJArc),其在内部在UNF的SPF动物设施内饲养
    3. OT-I小鼠(Hogquist等人,1994)
    4. 在SPF内部饲养的OT-IIxFoxp3-eGFP小鼠(称为OT-II(Barnden等人,1998,Wang等人,2008) UNIL的动物设施

  3. 抗体
    1. Va2和Vb5.1/5.2抗体(BD Biosciences)
    2. CD45 PE抗体(BD Biosciences)
    3. CD45.1 APC-eFluor780(eBioscience)
    4. CD45.2太平洋蓝(eBioscience)
    5. CD8 eFluor700(eBioscience)
    6. CD4 PE-德克萨斯红(eBioscience)
    7. 2.4G2(抗FcgRII单克隆抗体)

  4. 缓冲液和介质
    1. Dulbecco改良的Eagle's培养基(DMEM),高葡萄糖,GlutaMAX TM补充剂(Life Technologies,Gibco ,目录号:10566-016)
    2. 1 M HEPES(Life Technologies,Gibco ,目录号:15630-080)
    3. 青霉素 - 链霉素(5,000U/ml)(Life Technologies,Gibco ,目录号:15070063)
    4. 牛血清(FBS)。 (低内毒素的性能血清:合格,美国产)(Life Technologies,目录号:26140等)
    5. Geneticin选择性抗生素(G418硫酸盐,50mg/ml)(Life Technologies,Gibco ,目录号:15630-080)
    6. 0.25%胰蛋白酶-EDTA (1x)(酚红)(Life Technologies,Invitrogen TM ,目录号:25200056)
    7. 磷酸盐缓冲盐水(PBS)(Laboratorium Dr.Bichsel AG)
    8. 胶原酶,I型(Life Technologies,Gibco ,目录号:17018-029)
    9. DNAse我  (Roche Diagnostics,目录号:04536282001)
    10. UltraPure TM 0.5M EDTA(pH 8.0)(Life Technologies,Invitrogen TM,目录号:15575-020)
    11. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9418)
    12. 完成Dulbecco改良的Eagle培养基(cDMEM)(见Recipes)
    13. 肿瘤消化缓冲液(参见配方)
    14. 流缓冲区(参见配方)
    15. MACS缓冲区(参见配方)

    1. OVA257-264和OVA323-339肽
      注意:肽由UNIL的蛋白质和肽化学设备(PPCF)制造。

  5. 佐剂
    1. Poly(I:C)HMW  (tlrl-pic)and  咪喹莫特R837(tlrl-imq,InvivoGen)
    2. CpG-ODN 1826(Coley Pharmaceuticals,不再可用,CpG-ODN 1826,来自InvivoGen的tlrl-1826可以被取代)。
    3. 来自Quillaja saponaria(Brenntag Nordic A/S)的Quil A皂苷混合物

  6. 商业试剂
    1. CD45 MACS微珠,小鼠(Miltenyi Biotec,目录号:130-052-301)
      数据库在线提供: https://www.miltenyibiotec.com /~/media/Images/Products/Import/0001200/IM0001245.ashx
    2. LIVE/DEAD水性细胞染色剂(Life Technologies,Invitrogen TM ,目录号:L34957)

设备

  1. Falcon TM40μm细胞过滤器(蓝色)(Corning,目录号:352340)
  2. 1ml BD Tuberculin Syringe& 26g或27g×0.5"BD TM PrecisionGlide Needle(BD,目录号:3052111或305109)
  3. Falcon TM 50ml锥形离心管(Corning,目录号:352070)
  4. Falcon TM 15ml锥形离心管(Corning,目录号:352099)
  5. Falcon TM 6孔,未处理的平底组织培养物(Corning,目录号:351147)
  6. 解剖剪刀或手术刀刀片和细尖镊子或镊子
  7. 装有18g或20g×1"BD TM PrecisionGlide针(BD,目录号:305195或305175)的10ml BD TM注射器(目录号:309604)
  8. 血细胞计数器
  9. AutoMACS自动细胞分离器(Miltenyi Biotech)
  10. FACSCanto流式细胞仪(BD)

软件

  1. 适用于Mac的FlowJo软件,9版软件(TreeStar)
  2. Prism Graphpad软件
    注意:其用于进行单向Anova测试,结合Dunnet的后测试,用于每组3个样品的统计分析,每个实验。重复实验两次以获得统计鲁棒性。

程序

  1. 过继性细胞转移,肿瘤攻击和免疫
    1. 过继性细胞转移
      从CD45.2 + 的脾脏中分离抗原特异性CD8 + sup(OT-I)和CD4 + sup(OT-II) T细胞受体(TCR)转基因小鼠。在具有1ml结核菌素注射器柱塞的塑料末端的40μm细胞过滤器上破碎脾脏,并收集到50ml Falcon管中。通过用血细胞计数器计数确定总细胞数。通过用TCR Va2和Vb5.1/5.2抗体标记并在注射含有所需数量的抗原特异性T细胞的总脾细胞之前通过流式细胞术分析来测定脾中OT-I和OT-II细胞的频率(图1)。原始CD45.1受体小鼠在尾静脉中静脉内在200μlDMEM中接受1e6 OT-I细胞和3e6 OT-II细胞。
      注意:如果愿意,T细胞也可以通过眶内或腹膜内注射。
    2. 肿瘤挑战
      第二天用在左侧皮下注射的100μlPBS中的2e5 B16.OVA肿瘤细胞攻击小鼠。简言之,使用单手握持来约束小鼠。将约2/3的结核菌素注射器的针的长度以向上和侧向的角度插入接近后腿关节的皮肤的皮下层中。通过轻轻地移动皮肤下方的斜面(仅在髋关节周围)并确认其在松动的空间中,检查针的端部的正确位置。将肿瘤细胞缓慢注射到单个位置。小心取出针,用指尖轻轻按摩出口点,以防止肿瘤细胞逃逸
    3. 免疫
      1. 一周后,一旦肿瘤可触知,在100μlPBS中的10μgOVA sub 257-264和10μgOVA 323-339肽在皮下根据尾部用1ml结核菌素注射器。简言之,将小鼠约束在具有在末端开口的保持隧道中以接近尾部的基部。将结核菌素注射器的针的大约一半长度插入到刚好在尾部上方和侧面的皮肤的皮下层中。通过直接在皮肤下方观察斜面来确认正确的深度。在保持针入口点处的压力的同时缓慢注射疫苗。
        注意:如果应用正确的技术,液体泡沫应该在皮肤下可见。
      2. 肽单独注射或与50μg下列佐剂之一组合注射:CpG-ODN(CpG),HMW Poly(I:C),咪喹莫特或Quil A.

  2. 组织收获和加工
    1. 接种后一周,通过CO 2窒息,随后颈椎脱臼将小鼠安乐死。通过切割侧翼的皮肤暴露肿瘤,并且通过用细尖镊子小心地抓握并且使用尖锐的清除剪刀剪断周围的皮肤和结缔组织来切除肿瘤。然后将肿瘤置于预先称重的含有2ml PBS的15ml Falcon管中。
    2. 再次称重含有肿瘤的管,并减去管和缓冲液的初始重量以确定肿瘤质量
    3. 将大于500μg的肿瘤切割至约500μg用于处理,以避免MACS柱过载,并且在较大肿瘤中获得的细胞数目超过流式细胞术 分析
    4. 清除结缔组织的肿瘤,将每个肿瘤置于6孔板中的1ml新鲜的肿瘤消化缓冲液中。通过用剪刀剪切或通过用手术刀刀片重复切片以产生〜2mm或更小的片段来切割肿瘤。向每个孔中再加入9ml的Tumor消化缓冲液,并通过轻轻吸取与组织混合。
    5. 然后将肿瘤组织在37℃下孵育1小时以允许消化,旋转平板或在30分钟后向上和向下吸取悬浮液以混合。
    6. 在孵育结束时,将组织片通过具有大孔针(18-20号)的10ml注射器抽吸几次以破碎剩余组织并获得单细胞悬浮液。
    7. 将细胞在50ml cDMEM中洗涤2-3次,并以1,500rpm离心10分钟以除去过量的黑色素
    8. 使用血细胞计数器进行粗略的总细胞计数以确定所需的MACS珠子的量

  3. CD45 + 细胞选择
    1. 将肿瘤细胞转移到50ml Falcon管中,并根据制造商的方案将其重悬于90μlMACS缓冲液/10μL细胞中。
    2. 每10 7个细胞加入10μl抗CD45珠,并将细胞和珠混合物在冰上孵育15分钟,每5分钟颠倒混合。
    3. 然后将细胞在50ml MACS缓冲液中洗涤两次以除去多余的珠子
    4. 然后通过温和吸取将细胞重悬浮于500μlMACS缓冲液中,并通过40μm细胞过滤器以除去任何剩余的团块。
    5. CD45 +细胞通过使用磁性细胞分离(MACS)珠和AutoMACS自动细胞分离器的阳性选择来纯化。在通过使用POSSEL程序的自动MACS系统之前,通过低速涡旋混合细胞悬浮液。
    6. 在进行细胞的FACS染色之前,使用血细胞计数器计数阳性分数以确定总TIL数。
      注意:
      1. POSSEL程序使样品通过autoMACS,使用单次通过一个磁性柱,并且设计为通过从样品的阳性选择获得特定的细胞群体,其中细胞的正常至高频率表达感兴趣的抗原。
      2. 如果autoMACS机器不可用,则可以使用手动MACS柱(Miltenyi Biotech)。手动分离程序可在CD45 MACS微珠数据表(见G部分)中找到。
      3. 可以通过用CD45 PE抗体标记预分选和分选后阳性和阴性级分的小等分试样并通过流式细胞术分析(图2)来检查分选的纯度和产率。在前2个实验后,在阳性部分中产生> 90%CD45 +细胞,在全局FACS分析之前不再进行纯度测试。

  4. 流式细胞术
    1. 将〜5×10 6个细胞(或者如果获得小于该数目的整个样品)转移至96孔的u底板并用PBS洗涤。
    2. 将细胞在PBS中以1/250稀释度重悬于100μl活/死水细胞染色剂中,孵育20分钟,然后用PBS洗涤两次。
    3. 将来自产生2.4G2(抗FcgRII单克隆抗体)杂交瘤(室内生长)的25μl上清液加入样品中,并在冰上温育5-10分钟以抑制非特异性抗体结合。
    4. 在流动缓冲液中制备2x最终浓度的荧光抗体的混合物,并将25μl混合物加入细胞,而不首先除去2.4G2。
    5. 将细胞再培养20-30分钟,然后将板用200μl/孔的Flow缓冲液洗涤两次
    6. 将细胞重悬于200μl流动缓冲液中并保持在冰上直至分析。
    7. 使用LSR-II和FACSCanto流式细胞仪获得样品。
    8. 基于前向散射和侧向散射性质对淋巴细胞进行门控,并使用LIVE/DEAD Aqua细胞染色来排除死细胞。
    9. 使用FlowJo软件分析流量数据(图3)

  5. 多色染色板
    1. 活/死染料水族细胞染色
    2. CD45.1 APC-eFluor780,CD45.2 Pacific Blue,CD8 eFluor700,CD4 PE-Texas Red(任选地用Va2 APC和Vb5.1/5.2PE标记以更精确地鉴定TCR转基因T细胞)。
    3. Foxp3 eGFP(内源性表达)。
    4. 除了用于标记样品的多色染色混合物之外,对于每种荧光染料制备单染色(并且通过用于GFP单染色的尾静脉的缺刻来提取来自Foxp3-eGPF小鼠的血液)。
    5. BD Diva软件中的自动补偿选项用于补偿所有不同荧光染料之间的光谱重叠

代表数据



图1.在过继性T细胞转移(基于正向和侧向散射性质门控淋巴细胞群体)之前通过流式细胞术确定TCR转基因T细胞频率


图2. MACS阳性选择后的CD45细胞纯度(基于使用活/死染料水性细胞染色检测的前向散射高度对面积和活细胞的单个细胞首先门控)


图3.代表性流程图,其显示用于分析CD45 MACS富集的肿瘤样品中肿瘤浸润性淋巴细胞的渐进门控策略

食谱

  1. 完整媒体(cDMEM)
    DMEM
    5%FBS
    100U/ml青霉素& 100μg/ml链霉素 25mM Hepes
    55μM2-ME
  2. 肿瘤消化缓冲液
    DMEM
    4.4mg/ml胶原酶I
    10μg/ml DNase I
  3. 流缓冲区
    PBS
    2%FBS
  4. MACS缓冲区
    PBS
    1%BSA
    10 mM EDTA

致谢

以前发布的简称协议:Perret等人。(2013)。 这项工作得到了新西兰研究科学与技术基金会和Emma Muschamp基金会(R. Perret)和瑞士国家科学基金会(310030-130812和CRSII3_141879)和Medic基金会(P. Romero)的资助。 披露潜在的利益冲突:P. Romero是Immatics Biotechnologies,DC Prime,Matwin和巴斯德研究所(巴黎,法国)人类免疫中心的顾问/顾问委员会成员。 其他作者未公开任何潜在的利益冲突。

参考文献

  1. Barnden,M.J.,Allison,J.,Heath,W.R.and Carbone,F.R。(1998)。 使用基于cDNA的控制下的α和β链基因构建的转基因小鼠中的TCR表达缺陷的异源调节元件。 Immunol Cell Biol 76(1):34-40
  2. Fidler,I.J。(1975)。 恶性黑素瘤细胞的生物学行为与其在体内的存活相关。 Cancer Res 35(1):218-224
  3. Hogquist,K.A.,Jameson,S.C.,Heath,W.R.,Howard,J.L.,Bevan,M.J.and Carbone,F.R。(1994)。 T细胞受体拮抗剂肽诱导阳性选择。 细胞 76(1):17-27。
  4. Perret,R.,Sierro,S. R.,Botelho,N. K.,Corgnac,S.,Donda,A.和Romero,P.(2013)。 提高抗原特异性效应物与调节性T细胞比率的佐剂增强肿瘤免疫力。 Cancer Res 73(22):6597-6608。
  5. Schuler,P.,Contassot,E.,Irla,M.,Hugues,S.,Preynat-Seauve,O.,Beermann,F.,Donda,A.,French,L.E.and Huard, 外周淋巴结中黑素细胞相关抗原的直接呈递诱导细胞毒性CD8 + sup> T cells。 Cancer Res 68(20):8410-8418。
  6. Wang,Y.,Kissenpfennig,A.,Mingueneau,M.,Richelme,S.,Perrin,P.,Chevrier,S.,Genton,C.,Lucas,B.,DiSanto,JP,Acha-Orbea, ,Malissen,B。和Malissen,M。(2008)。 Lat2136F突变小鼠的Th2淋巴增殖性疾病独立于TCR-MHC参与而展开,并且对于 Foxp3 + 调节性T细胞。 J Immunol 180(3):1565-1575。
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引用:Perret, R., Sierro, S. R., Botelho, N. K., Corgnac, S., Donda, A. and Romero, P. (2014). Analysis of Tumor-infiltrating Lymphocytes Following CD45 Enrichment . Bio-protocol 4(16): e1218. DOI: 10.21769/BioProtoc.1218.
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