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Adoptive Transfer of Tumor Expanded Regulatory T Cells (Tregs)
肿瘤扩增调节性T细胞的过继转移   

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Abstract

Regulatory T cells (Tregs), a subset of CD4+CD25+ T cells, infiltrate tumors and suppress antitumor activity of effector T and NK cells. Depletion of Tregs by anti CD25+ antibodies has been shown to reduce tumor growth and metastasis (Olkhanud et al., 2009). Conversely, adoptive transfer of Tregs induced immune suppression and promoted tumor growth (Smyth et al., 2006; Janakiram et al., 2015). We have adoptively transferred Tregs to evaluate their immunosuppressive function in vivo. Our study (Vences-Catalan et al., 2015) compared the immunosuppressive efficacy of Tregs derived from tumor-bearing wild type to those of CD81KO mice. The following protocol could be adapted to any other source of Tregs.

Lymph node or splenic tumor-induced Tregs are isolated and purified by a two-step procedure using CD4+CD25+ regulatory T cell isolation kit from MACS Miltenyi Biotec. First, CD4+ T cells are enriched by negative selection, followed by positive selection of CD25+ T cells. Tumor-induced purified Tregs (CD3+CD4+CD25+FoxP3+) are then co-injected subcutaneously together with tumor cells into naïve mice (Winn assay) (Winn, 1960). Tregs could also be injected intravenously once or several times, according to the research needs. The effect of the adoptively transferred Tregs on tumor growth is then measured by caliper or by in vivo imaging techniques.

Keywords: Tregs(调节性T细胞), Adoptive transfer(过继转移), Tumor(肿瘤), Immune suppression(免疫抑制)

Materials and Reagents

  1. 70 μm cell strainer (Corning, Falcon®, catalog number: 352350 )
  2. 1 ml syringe (BD, catalog number: 309659 )
  3. 30 G x 1/2 needle (BD, catalog number: 305106 )
  4. LD columns (Miltenyi Biotec, catalog number: 130-042-901 )
  5. MS columns (Miltenyi Biotec, catalog number: 130-042-201 )
  6. 15 and 50 ml polypropylene conical tubes (Corning, Falcon®, catalog number: 352096 and 352070 )
  7. Mice
    Note: We use 6-8 weeks old female Balb/c or C57BL6 mice, but the protocol is applicable to any mouse strain as long as both donor and recipient mice are from the same genetic background.
  8. Cells
    Note: We use breast cancer cell lines 4T1 or E0771 syngeneic to Balb/c or C57Bl6 respectively.
  9. Fetal calf serum (FCS) (GE life sciences, HyCloneTM, FetalClone®III, catalog numer: SH30109.03 )
  10. Penicillin-Streptomycin (Pen-Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-22 )
    Note: 5 ml of this solution was used in 500 ml of RPMI 1640 media.
  11. PBS (Corning, Cellgro, catalog number: 21-031-CV )
  12. ACK buffer (Quality Biological, catalog number: 118-156-101 )
  13. CD4+CD25+ Regulatory T cell Isolation kit (Miltenyi Biotec, catalog number: 130-091-041 )
    Components of the kit:
    1. 1 ml CD4+CD25+ Regulatory T cell Biotin-antibody cocktail
    2. 2 ml Anti-biotin Microbeads 
    3. 1 ml CD25-PE 
    4. 1 ml Anti-PE Microbeads
  14. RPMI 1640 (Corning, Cellgro, catalog number: 10-040-CV )
  15. Bovine serum albumin (BSA) (Sigma Aldrich, catalog number: A9647-500G )
    Note: A solution of 0.5% in PBS was used. 
  16. EDTA (Sigma Aldrich, catalog number: E-5134 )
    Note: A solution of 2 mM in PBS was used.
  17. Optional: CD3 FITC (clone:145-2C11) (BD Pharmingen)
  18. Optional: CD25 PE (clone:PC61.5) (BD Pharmingen) 
  19. Optional: CD4 PerCP (clone:H129.19/RM4-5) (BD Pharmingen)
  20. Optional: FoxP3 APC (clone:FJK16s) (BD Pharmingen)
  21. MACS Buffer (see Recipes)

Equipment

  1. MidiMACS separator (Miltenyi Biotec, catalog number: 130-042-302 )
  2. MiniMACS separator (Miltenyi Biotec, catalog number: 130-042-102 )
  3. Centrifuge (Eppendorf, model: 5810 R )

Procedure

  1. Induction of regulatory T cells
    4T1 or E0771 cells were grown in RPM media, supplemented with 10% FCS and 1% Pen-Strep. Inject subcutaneously 1-5 x 104 4T1 or 0.5-1 x 106 E0771 cells resuspended in PBS (90% or greater viability) into either Balb/c or C57BL6 mice, respectively (an average of 3-5 mice are needed to obtain around 1-5 x 106 Tregs, if more Tregs are needed inject more mice).

  2. Sample preparation for Treg purification
    After 12-16 days, euthanize the mice by cervical dislocation (AVMA Guidelines for the Euthanasia of Animals: 2013 Edition) and remove lymph nodes (inguinal and axillary) (Mathieu et al., 2012) and/or spleens. Prepare single-cell suspension by mechanical disruption of the tumor (make sure to work with single cell suspension, by passing through a 70 μm cell strainer).
    Optional: If red blood cells are present, lyse with ACK buffer, although it is not necessary as the kit contains anti Ter-119 antibody to positively deplete red blood cells.
    Note: Work fast, keep cells cold and use pre-cooled solutions. This will prevent capping of antibodies on the cell surface and will reduce non-specific cell labeling.

  3. Magnetic labeling of non-CD4+ cells and fluorescent labeling of CD25+ cells
    We follow exactly the protocol provided by the manufacturer to purify CD4+CD25+ Regulatory T cells. Volumes for magnetic labeling given below are for starting cell number of 107 leukocytes. When working with fewer than 107 leukocytes use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly.
    1. Determine the number of leukocytes.
    2. Centrifuge cells at 300 x g for 10 min at room temperature (RT). Aspirate supernatant completely.
    3. Resuspend cell pellet in 40 μl of MACS buffer per 107 total cells.
    4. Add 10 μl of Biotin-Antibody cocktail per 107 total cells (this cocktail contains anti-mouse antibodies against: CD8a, CD11b, CD45R, CD49b and Ter-119).
    5. Mix well by pipetting up and down, and refrigerate for 10 min (4-8 °C).
    6. Add 30 μl of MACS buffer, 20 μl of anti-biotin Microbeads and 10 μl of CD25-PE antibody per 107 total cells.
    7. Mix well and refrigerate for an additional 15 min in the dark (4-8 °C).
    8. Wash cells by adding 1-2 ml of buffer per 107 total cells and centrifuge at 300 x g for 10 min RT. Aspirate supernatant completely.
    9. Resuspend cell pellet in 500 μl of MACS buffer (up to 1.25 x 108 cells can be suspended in this volume according to the manufacturer).
    10. Proceed to magnetic separation.


      Figure 1. MACS midi system for cell purification

  4. Depletion with LD column
    1. Place LD column in the magnetic field of a midi MACS separator (Figure 1).
    2. Prepare column by rinsing with 2 ml of MACS buffer.
    3. Apply cell suspension to the column.
    4. Collect unlabeled cells which pass through and wash column 2 times by pipetting 1 ml of MACS buffer. Perform washing steps by adding MACS buffer successively once the column reservoir is empty. Collect total effluent. This is the unlabeled CD4+ T cell fraction.
    5. Proceed for the enrichment of CD4+CD25+ T cells.

  5. Magnetic labeling of CD25+ cells
    Volumes for magnetic labeling given below are for up to 107 initial starting cell number. For larger initial cell numbers, scale up accordingly.
    1. Centrifuge isolated CD4+ T cells at 300 x g for 10 min. Aspirate supernatant completely.
    2. Resuspend cell pellet in 90 μl of MACS buffer.
    3. Add 10 μl of anti-PE Microbeads.
    4. Mix well and refrigerate for 15 min in the dark (4-8 °C).
    5. Wash cells by adding 1-2 ml of MACS buffer and centrifuge at 300 x g for 10 min. Aspirate supernatant completely.
    6. Resuspend up to 108 cells in 500 μl of MACS buffer.
    7. Proceed to magnetic separation.

  6. Magnetic separation: Positive selection of CD4+CD25+ regulatory T cells
    1. Place MS column in the magnetic field of a suitable MACS separator.
    2. Prepare column by rinsing with 500 μl of MACS buffer.
    3. Apply cell suspension onto the column.
    4. Let cells pass through and wash column 3 times with 500 μl of MACS buffer. Perform washing steps by adding MACS buffer three times, once the column reservoir is empty.
    5. Remove column from the separator and place it on a suitable collection tube.
    6. Pipette 1 ml of MACS buffer on to the column. Immediately flush out the magnetically labeled cells (CD4+CD25+) by firmly pushing the plunger into the column.
    7. Optional: To achieve highest purities, repeat magnetic separation with a consecutive column run.

  7. Co-injection of Tregs with 4T1 or E0771 tumor cells
    1. Determine the cell number of Tregs and tumor cells needed for injection.
    2. Inject approximately 100-200 μl of PBS containing a mixture purified Tregs with tumor cells subcutaneously close to the mammary fat pad. 
    3. Follow tumor growth by caliper measurements.

Representative data


Figure 2. Representative Treg purification

Notes

  1. Tregs purity varies from 85-95% (See Figure 2). 
  2. On our experiments we transferred 5 x 104 4T1 cells together with 1 x 106 Tregs or 0.5 x 106 E0771 cells with 1 x 106 Tregs.

Recipes

  1. MACS Buffer
    PBS supplemented with 0.5% BSA and 2 mM EDTA
    Keep buffer cold (4-8 °C)

Acknowledgments

This work was supported by the Translational Cancer Award from Stanford Cancer Institute and the Breast Cancer Research program from the Department of Defense grant W81XWH-14-1-0397. Adapted from (Winn, 1960).

References

  1. Janakiram, N. B., Mohammed, A., Bryant, T., Brewer, M., Biddick, L., Lightfoot, S., Lang, M. L. and Rao, C. V. (2015). Adoptive transfer of regulatory T cells promotes intestinal tumorigenesis and is associated with decreased NK cells and IL-22 binding protein. Mol Carcinog 54(10): 986-998. 
  2. Mathieu, M. and Labrecque, N. (2012). Murine superficial lymph node surgery. J Vis Exp (63): e3444.
  3. Olkhanud, P. B., Baatar, D., Bodogai, M., Hakim, F., Gress, R., Anderson, R. L., Deng, J., Xu, M., Briest, S. and Biragyn, A. (2009). Breast cancer lung metastasis requires expression of chemokine receptor CCR4 and regulatory T cells. Cancer Res 69(14): 5996-6004.
  4. Smyth, M. J., Teng, M. W., Swann, J., Kyparissoudis, K., Godfrey, D. I. and Hayakawa, Y. (2006). CD4+CD25+ T regulatory cells suppress NK cell-mediated immunotherapy of cancer. J Immunol 176(3): 1582-1587. 
  5. Vences-Catalan, F., Rajapaksa, R., Srivastava, M. K., Marabelle, A., Kuo, C. C., Levy, R. and Levy, S. (2015). Tetraspanin CD81 promotes tumor growth and metastasis by modulating the functions of T regulatory and myeloid-derived suppressor cells. Cancer Res 75(21): 4517-4526.
  6. Winn, H. J. (1960). The immune response and the homograft reaction. Natl Cancer Inst Monogr 2: 113-138.

简介

调节性T细胞(Treg)(CD4 + CD25 + T细胞的子集)浸润肿瘤并抑制效应T和NK细胞的抗肿瘤活性。已显示抗CD25 sup/+抗体减少Treg减少肿瘤生长和转移(Olkhanud等人,2009)。相反,Treg的过继转移诱导免疫抑制并促进肿瘤生长(Smyth等人,2006; Janakiram等人,2015)。我们已经过继转移Treg以评价它们在体内的免疫抑制功能。我们的研究(Vences-Catalan等人,2015)比较了源自荷瘤野生型的Treg与CD81KO小鼠的免疫抑制功效。以下协议可以适用于任何其他来源的Tregs。
 通过使用来自MACS Miltenyi Biotec的CD4 + CD25 +调节性T细胞分离试剂盒的两步程序分离和纯化淋巴结或脾肿瘤诱导的Treg。首先,通过阴性选择,然后CD25阳性选择+ T细胞富集CD4 + T细胞。然后将肿瘤诱导的纯化的Treg(CD3 + CD4 CD25 + FoxP3 + 与肿瘤细胞转化为幼稚小鼠(Winn测定)(Winn,1960)。根据研究需要,Treg也可以静脉内注射一次或几次。然后通过测径器或通过体内成像技术测量过继转移的Treg对肿瘤生长的影响。


关键字:调节性T细胞, 过继转移, 肿瘤, 免疫抑制

材料和试剂

  1. 70μM细胞过滤器(Corning,Falcon ,目录号:352350)
  2. 1ml注射器(BD,目录号:309659)
  3. 30G×1/2针(BD,目录号:305106)
  4. LD柱(Miltenyi biotec,目录号:130-042-901)
  5. MS柱(Miltenyi biotec,目录号:130-042-201)
  6. 15和50ml聚丙烯锥形管(Corning,Falcon ,目录号:352096和352070)
  7. 小鼠
    注意:我们使用6-8周龄的雌性Balb/c或C57BL6小鼠,但是该方案适用于任何小鼠品系,只要供体和受体小鼠来自相同的遗传背景。
  8. 单元格
    注意:我们分别使用乳腺癌细胞系4T1或E0771与Balb/c或C57Bl6同源。
  9. 胎牛血清(FCS)(GE life sciences,HyClone ,FetalClone III,目录号:SH30109.03)
  10. 青霉素 - 链霉素(Pen-Strep)(Thermo Fisher Scientific,Gibco TM,目录号:15140-22)
    注意:将5ml该溶液用于500ml RPMI 1640培养基中。
  11. PBS(Corning,Cellgro,目录号:21-031-CV)
  12. ACK缓冲液(Quality Biological,目录号:118-156-101)
  13. CD4 CD25 + 调节性T细胞分离试剂盒(Miltenyi Biotec,目录号:130-091-041)
    套件的组件:
    1. 1ml CD4 +细胞生物素 - 抗体混合物
    2. 2 ml抗生物素微珠
    3. 1ml CD25-PE
    4. 1 ml抗PE微珠
  14. RPMI 1640(Corning,Cellgro,目录号:10-040-CV)
  15. 牛血清白蛋白(BSA)(Sigma Aldrich,目录号:A9647-500G)
    注意:使用0.5%的PBS溶液。
  16. EDTA(Sigma Aldrich,目录号:E-5134) 注意:使用2mM的PBS溶液。
  17. 任选:CD3FITC(克隆:145-2C11)(BD Pharmingen)
  18. 任选:CD25 PE(克隆:PC61.5)(BD Pharmingen)
  19. 可选:CD4 PerCP(克隆:H129.19/RM4-5)(BD Pharmingen)
  20. 可选:FoxP3 APC(克隆:FJK16s)(BD Pharmingen)
  21. MACS缓冲区(参见配方)

设备

  1. MidiMACS分离器(Miltenyi Biotec,目录号:130-042-302)
  2. MiniMACS分离器(Miltenyi Biotec,目录号:130-042-102)
  3. 离心机(Eppendorf,型号:5810R)

程序

  1. 诱导调节性T细胞
    4T1或E0771细胞在补充有10%FCS和1%Pen-Strep的RPM培养基中生长。 皮下注射重悬于PBS(90%或更高的存活力)中的1-5×10 4个4T1或0.5-1×10 6个E0771细胞到Balb/c或C57BL6小鼠中 (如果需要更多的Treg注射更多的小鼠,则平均需要3-5只小鼠来获得约1-5×10 6个 Treg)。

  2. 用于Treg纯化的样品制备
    在12-16天后,通过颈部脱臼(AVMA动物安乐死指南:2013版)安乐死小鼠,并除去淋巴结(腹股沟和腋窝)(Mathieu等人,2012)和/或脾。通过机械破坏肿瘤制备单细胞悬浮液(确保使用单细胞悬液,通过通过70微米的细胞过滤器)。 可选:如果存在红细胞,用ACK缓冲液溶解,但不是必需的,因为该试剂盒含有抗Ter-119抗体以正性消耗红细胞。
    注意:工作速度快,保持细胞冷,并使用预冷解决方案。这将防止细胞表面的抗体上覆,并减少非特异性细胞标记。

  3. 非CD4 + 细胞的磁性标记和CD25 + 细胞的荧光标记
    我们完全遵循制造商提供的方案以纯化CD4 + CD25 + sup/+调节性T细胞。下面给出的磁性标记的体积用于起始细胞数为10 7个白细胞。当使用少于10个的白细胞工作时,使用与所示相同的体积。当使用较高的细胞数量时,相应地扩大所有试剂量和总量
    1. 确定白细胞数。
    2. 在室温(RT)下将细胞在300×g离心10分钟。完全吸出上清液。
    3. 重悬细胞沉淀在40微升MACS缓冲区每10 总细胞。
    4. 每10μL总细胞加入10μl生物素 - 抗体混合物(该混合物含有针对CD8a,CD11b,CD45R,CD49b和Ter-119的抗小鼠抗体)。
    5. 通过上下吹吸混匀,冷藏10分钟(4-8℃)。
    6. 每10 7个总细胞加入30μl的MACS缓冲液,20μl的抗生物素微珠和10μl的CD25-PE抗体。
    7. 充分混合并在黑暗中(4-8℃)冷藏另外15分钟。
    8. 通过每10 7个总细胞加入1-2ml缓冲液洗涤细胞,并在300×g离心10分钟RT。 完全吸出上清液。
    9. 将悬浮细胞沉淀在500μlMACS缓冲液中(根据制造商,可将高达1.25×10 8个细胞悬浮在该体积中)。
    10. 继续进行磁分离。


      图1.细胞纯化的MACS midi系统

  4. 用LD柱消耗
    1. 将LD柱置于midi MACS分离器的磁场中(图1)。
    2. 通过用2ml MACS缓冲液冲洗制备柱。
    3. 将细胞悬液应用于色谱柱。
    4. 收集未标记的细胞,其通过并通过吸取1ml MACS缓冲液洗涤柱2次。 当柱容器为空时,连续添加MACS缓冲液执行清洗步骤。 收集总流出物。 这是未标记的CD4 + T细胞部分。
    5. 继续进行CD4 + CD25 + T细胞的富集。

  5. CD25 + 细胞的磁性标记
    下面给出的磁性标记体积是最多10个 7 初始起始细胞数。 对于较大的初始单元格数量,相应地按比例增加
    1. 离心分离的CD4 + T细胞在300×g离心10分钟。 完全吸出上清液。
    2. 重悬细胞沉淀在90微升的MACS缓冲液。
    3. 加入10μl的抗PE微珠。
    4. 充分混合并在黑暗中(4-8℃)冷藏15分钟。
    5. 通过加入1-2ml MACS缓冲液洗涤细胞,并在300×g离心10分钟。 完全吸出上清液。
    6. 在500μlMACS缓冲液中重悬最多10个细胞。
    7. 继续进行磁分离。

  6. 磁性分离:CD4 + CD25 + sup/+调节性T细胞的阳性选择
    1. 将MS柱置于合适的MACS分离器的磁场中。
    2. 通过用500μlMACS缓冲液漂洗来制备柱。
    3. 将细胞悬液应用于色谱柱。
    4. 让细胞通过,用500μlMACS缓冲液洗涤柱3次。 执行洗涤步骤,通过添加MACS缓冲液三次,一旦柱容器是空的。
    5. 从分离器中取出色谱柱,并将其放在合适的收集管上。
    6. 移取1ml的MACS缓冲液到柱上。 通过将柱塞牢固地推入柱子,立即冲出磁性标记的细胞(CD4 + CD25 + )。
    7. 可选:要达到最高纯度,使用连续柱运行重复磁选分离
  7. Treg与4T1或E0771肿瘤细胞的共注射
    1. 确定注射所需的Treg和肿瘤细胞的细胞数。
    2. 注射约100-200微升含有混合物纯化的Treg的PBS,其中肿瘤细胞皮下接近乳腺脂肪垫。
    3. 通过卡尺测量跟踪肿瘤生长。

代表数据


图2.代表性的Treg纯化

笔记

  1. Tregs纯度从85-95%变化(参见图2)。
  2. 在我们的实验中,我们将5×10 4个4T1细胞与1×10 6个Treg或0.5×10 6个E0771细胞一起转移到1×10 6个 6 Tregs。

食谱

  1. MACS缓冲区
    补充有0.5%BSA和2mM EDTA的PBS 保持缓冲液冷(4-8°C)

致谢

这项工作得到斯坦福癌症研究所的翻译癌症奖和国防部授予的乳腺癌研究计划W81XWH-14-1-0397的支持。改编自(Winn,1960)。

参考文献

  1. Janakiram,N.B.,Mohammed,A.,Bryant,T.,Brewer,M.,Biddick,L.,Lightfoot,S.,Lang,M.L.and Rao, 调节性T细胞的过继转移促进肠道肿瘤发生,并与减少NK细胞和IL-22结合蛋白。 Mol Carcinog 54(10):986-998。
  2. Mathieu,M。和Labrecque,N。(2012)。 鼠表面淋巴结手术。 Vis Exp (63):e3444。
  3. Olghanud,PB,Baatar,D.,Bodogai,M.,Hakim,F.,Gress,R.,Anderson,RL,Deng,J.,Xu,M.,Briest,S。和Biragyn, 。 乳腺癌肺转移需要趋化因子受体CCR4和调节性T细胞的表达。 Cancer Res 69(14):5996-6004。
  4. Smyth,M.J.,Teng,M.W.,Swann,J.,Kyparissoudis,K.,Godfrey,D.I。和Hayakawa,Y。(2006)。 CD4 + CD25 + 176(3):1582-1587。 T调节细胞抑制NK细胞介导的癌症免疫治疗。
  5. Vences-Catalan,F.,Rajapaksa,R.,Srivastava,M.K.,Marabelle,A.,Kuo,C.C.,Levy,R.and Levy,S。 Tetraspanin CD81通过调节T调节功能来促进肿瘤生长和转移 和髓样衍生的抑制细胞。 Cancer Res 75(21):4517-4526。
  6. Winn,H.J。(1960)。 免疫反应和同种异体移植反应 Natl Cancer Inst Monogr 2:113-138。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Vences-Catalán, F. and Levy, S. (2016). Adoptive Transfer of Tumor Expanded Regulatory T Cells (Tregs). Bio-protocol 6(16): e1899. DOI: 10.21769/BioProtoc.1899.
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