T Cell Transfer Model of Colitis

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Nature Communications
May 2015



Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC) is mainly caused by disordered immune regulation and dysregulated intestinal microbiota. Here we present the T cell transfer model which has extensively used in many studies to identify the regulatory T cell function in gut inflammation. Naïve T cells only or together with Treg cells isolated from different donors were transferred into immunodeficient Rag1-/- mice and the disease progression was assessed by the loss of body weight and the scoring analysis. This model provides a reliable work system for the study of gut inflammation.

Keywords: Induced colitis (诱导的结肠炎), Mouse model (小鼠模型), T cells (T细胞), Gut inflammation (肠道炎症), Rag1-/- (RAG1 /—)

Materials and Reagents

  1. 15 ml polypropylene conical tubes (Corning, catalog number: 430790 )
  2. 50 ml polypropylene conical tubes (Corning, catalog number: 430828 )
  3. LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
  4. 6-8 weeks old C57BL/6J WT mice used for donors (males)
  5. 6-8 weeks old Immunodeficient RAG1−/− mice (males) (The Jackson Laboratory, Stock number: 00 2216 )
  6. 10 cm petri dish (Corning, catalog number: 430167 )
  7. 10 ml syringe (BD Biosciences, catalog number: 301604 )
  8. 27 gauge needle (BD Biosciences, catalog number: 305109 )
  9. 40 μm nylon cell strainer (Corning, Falcon®, catalog number: 352340 )
  10. 70 μm cell filter (Corning, Falcon®, catalog number: 352350 )
  11. Polystyrene round-bottom flow tubes (Corning, Falcon®, catalog number: 352054 )
  12. FBS (Thermo Fisher Scientific, GibcoTM, catalog number: 10100-147 )
  13. RPMI media 1640 (Thermo Fisher Scientific, GibcoTM, catalog number: 11875-093 )
  14. ACK lysing buffer for erythrocyte lysis (Thermo Fisher Scientific, GibcoTM, catalog number: A10492-1 )
  15. Mouse CD4 Negative Isolation Kit (Miltenyi Biotec, catalog number: 130-095-248 )
  16. Fluorescently labeled antibodies:
    1. anti-CD25-PE (BioLegend, catalog number: 102012 )
    2. anti-CD4-PE-Cy7 (BD Biosciences, catalog number: 563933 )
    3. anti-CD44-APC (BD Biosciences, catalog number: 559250 )
    4. anti-CD62L-FITC (BD Biosciences, catalog number: 553150 )
  17. 37% Formaldehyde (formalin) Solution (Avantor Performance Materials, J.T.Baker®, catalog number: 2016 )
  18. FACS buffer (see Recipes)


  1. Plain glass microscope slides (Omano, catalog number: OMSK-50PL )
  2. Hemocytometer
  3. Light microscope
  4. Top-loading balance
  5. Centrifuge
  6. Flow cytometry (BD Biosciences, model: ARIA III )


  1. Enrichment for CD4+ T cells using magnetic beads
    1. Euthanize donor WT mice.
    2. Harvesting spleen from mouse.
    3. Place the dissected organs in 10 cm petri dish with 10 ml PBS + 2% FBS. Use two pieces of sterile frosted glass (keep in -20 °C for 1 h in advance) slides to gently grind the tissues. For detail grinding procedure, please refer to http://www.bio-protocol.org/e834.
    4. Place 70 μm cell filter on top of a 50 ml tube and transfer fluid from dish to filter.
    5. Spin spleen suspension for 5 min at 4 °C, 300 x g (Note: This should be the centrifugation condition for all steps.). Discard supernatant by aspiration with care not to disturb the pellets.
    6. Add 4 ml ACK lysis buffer and incubate at room temperature for 3 min. Add another 10 ml PBS + 2% FBS.
    7. Place 40 μm cell filter on top of a 50 ml tube and transfer fluid from 50 ml tube to filter. Spin for 5 min and discard supernatant by pouring off.
    8. Wash cells 2 times with 10 ml PBS + 2% FBS. Resuspend cells with PBS + 2% FBS buffer. Take an aliquot to count cells. Add 10 μl of cell suspension to 90 μl of Trypan blue solution (1:10 dilution) and count cells using a hemocytometer.
    9. Enrich CD4+ T cells by using the CD4+ T Cell Isolation Kit from Miltenyi Biotec.
    10. Following the negative selection protocol, pellet cells by centrifugation at 300 x g at 4 °C for 10 min.
    11. Discard most of the supernatant by aspiration; resuspend the cell pellet in 1 ml PBS + 2% FBS buffer by carefully pipetting up and down several times. Take an aliquot to count cells. Add 10 μl of cell suspension to 90 μl of Trypan blue solution (1:10 dilution) and count cells using a hemocytometer.

  2. Cell surface antigen staining for cell sorting
    1. We transfer appropriate number of cells into FACS tube and spin at 300-400 x g for 5 min.
    2. Prepare antibody cocktail containing anti-CD4-PE-Cy7, anti-CD25-PE, anti-CD44-APC, anti-CD62L-FITC antibodies diluted in 1x PBS with 2% FBS to optimal concentration (1:100). For every million cells, we usually add 100 μl 1x PBS with 2% FBS buffer with diluted antibody for incubation. According to the ratio and cells to be stained, prepare an antibody/PBS master mix.
    3. Resuspend the cells in 1x PBS with 2% FBS to obtain concentration of 25-50 x 106 cells/ml. The concentration of cells is dependent on the specific cell sorter to be used. Add the above mixture to cell pellet. Vortex to mix well. Stain in 4 °C for 15 min.
    4. Wash cells twice by filling tube with 1x PBS + 2% FBS. Spin cells down at 300 x g for 5 min at 4 °C. Pour off liquid.
    5. If debris or clumps are visible it is necessary to pass cells through a 40 μm cell strainer prior to cell sorting
    6. Protected cells from light and placed on ice prior to sorting.
    7. Sort naive T cells (CD4+CD25-CD44loCD62Lhi) and Treg populations (CD4+CD25hi) into RPMI media 1640 with 2% FBS.

  3. Injection of RAG1−/− mice with flow-purified naïve T cells and Treg cells
    1. After sorting, fill the collection tubes containing sorted cells with cold 1x PBS to collect cells in the walls and pellet by centrifugation as described above. Always keep cells on ice to avoid clump in each step.
    2. Wash cells 2 times with cold 1x PBS. This step is intended to deplete trace FBS. Resuspend cells very gently and prepare a single-cell suspension with PBS only and placed the cells on ice prior to injection. Take an aliquot to count cells. Add 10 μl of cell suspension to 90 μl of Trypan blue solution (1:10 dilution) and take 10 μl diluted cell suspension out to count cells using a hemocytometer.
    3. After counting, dilute cells with cold 1x PBS and adjust into a final cell concentration of 2 x 106/ml. Keep injection tubes on ice prior to injection.
    4. Mix cells for different groups. For every mouse, either 4 x 105 naïve T cells alone (200 μl) or 4 x 105 naïve T (200 μl) together with 2 x 105 Treg cells (100 μl) are mixed.
    5. Weigh recipient mice using a top-loading balance and record the weight. Prior to the injection of the mice gently invert the tube(s) to mix cells.
    6. Restrain the mouse use a tail access mouse restrainer. For detail restrainer picture, please refer to: http://www.stoeltingco.com/neuroscience/misc/restrainers.html. Rotate the tail slightly to visualize vein. Very slowly draw the cells into 1-ml syringe with the 27 gauge needle attached. Insert needle into the vein at a slight angle and inject all cells in.

  4. Monitoring disease progression and assessment of colitis
    Monitor disease progression by weighing each mouse twice per week using a top-loading balance. The mice get typically slow but progressive weight loss in the following 5-7 wk, accompanied by appearance of loose stools and diarrhea as well as a “hunched-over” appearance.
    Note: Some mice do not develop significant loose stools and diarrhea. However, these mice may still develop significant intestinal inflammation, which will be visible upon H&E staining.
    1. When mice lose about 15-20% of their original body weight or at about 40 d post injection, the animals should be euthanized and assessed for evidence of colitis.
    2. Carefully excise the entire colon starting from the anus and cutting it free of the mesentery from below the cecum to just above the anal opening. Be careful not to stretch of the colon.
    3. Place the colon on a plastic board, measure and record its length. Then clean it of fecal material by injecting PBS. Weigh the colonic tissue
    4. Colonic length and weight may be recorded to calculate length-to-weight ratio, which has been shown to correlate well with blinded histopathological scores.
    5. For histological processing and blinded histopathological scoring, a small (0.5 cm) piece from proximal and distal portions of the colon is obtained. These pieces of tissue are then placed in 10% PBS-formalin and allowed to fix for at least 24 h at room temperature prior to paraffin embedding.
    6. Embed fixed tissue into paraffin blocks. Section the paraffin-embedded samples produce 4-μm sections. Mount three sections on a microscopic glass slide, stain the sections with hematoxylin and eosin.
    7. Perform blinded histopathological analysis using the following scoring criteria:
      1. Degree of inflammation in lamina propria (score 0-3)
      2. Goblet cell loss (score 0-2)
      3. Abnormal crypts (score 0-3)
      4. Presence of crypt abscesses (score 0-1)
      5. Mucosal erosion and ulceration (score 0-3)
      6. Submucosal spread to transmural involvement (score 0-3)
      7. Number of neutrophils counted at 40x magnification (score 0-4)
      Total histopathological score is calculated by combining the scores for each of the seven parameters for a maximum score of 17.

Representative data

Figure 1. Schematic illustration of naïve T cell markers. Splenic cells were resuspended and stained with antibody cocktail containing anti-CD4-PE-Cy7, anti-CD25-PE, anti-CD44-APC, anti-CD62L-FITC antibodies for in 4 °C for 15 min. Naive T cells (CD4+CD25-CD44loCD62Lhi) were gated as above.

Figure 2. Schematic illustration of how T cell transfer model of colitis was investigated. Each Rag1–/– recipient mouse was transferred with 4 x 105 CD4+CD25-CD44loCD62Lhi naive T cells isolated from wild-type mice, and co-transferred with 2 x 105 CD4+CD25hi Treg cells isolated from WT (b) The weight loss of the mice in (a) was monitored every four days for 40 days from the day after injection. n = 6 mice for each group. (c) Representative H&E staining of colon sections from untreated WT mice (control), Rag1–/– mice transferred with WT naive T cells (naive), Rag1–/– mice co-transferred with WT naive T cells and WT Treg cells (naive + WT Treg (original magnification, 20x).


  1. Transfer results in no colitis development. Possible causes:
    1. Low naive T cell numbers transferred.
      Cell viability is another critical factor. Make sure the cells are always maintained on ice. It is important to assess the cell viability after the sorting is completed and only >90% viability is acceptable for transfer.
  2. It is important to remember that this model depends on many additional factors apart from the viability of naive T cells, sufficient naive T cells, and good animal procedures. These include the cleanliness of the animal house and the intact intestinal flora in recipient mice.


  1. FACS buffer
    2% FBS in PBS, keep cold for staining.


This protocol was modified from our previous work by Pan et al. (2015). This work is supported by grants from 973 program (2014CB541902, 2014CB541901), National Natural Science Foundation of China (81230072, 81025016, 31370880), the Key Research Program of the Chinese Academy of Sciences (KSZD-EW-Z-003-3), as well as Chinese Ministry of Health (201202008) and the Program of the Shanghai Commission of Science and Technology (12431900703, 12JC1406000, 12ZR1435900).


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  2. Pan, W., Zhu, S., Dai, D., Liu, Z., Li, D., Li, B., Gagliani, N., Zheng, Y., Tang, Y., Weirauch, M. T., Chen, X., Zhu, W., Wang, Y., Chen, B., Qian, Y., Chen, Y., Fang, J., Herbst, R., Richman, L., Jallal, B., Harley, J. B., Flavell, R. A., Yao, Y. and Shen, N. (2015). MiR-125a targets effector programs to stabilize Treg-mediated immune homeostasis. Nat Commun 6: 7096.
  3. Strober, W., Fuss, I. J. and Blumberg, R. S. (2002). The immunology of mucosal models of inflammation. Annu Rev Immunol 20: 495-549.
  4. Strober, W., Fuss, I. and Mannon, P. (2007). The fundamental basis of inflammatory bowel disease. J Clin Invest 117(3): 514-521.
  5. Xavier, R. J. and Podolsky, D. K. (2007). Unravelling the pathogenesis of inflammatory bowel disease. Nature 448(7152): 427-434.


炎症性肠病(IBD),包括克罗恩病(CD)和溃疡性结肠炎(UC),主要由无序的免疫调节和肠道微生物群失调引起。 在这里我们提出的T细胞转移模型已广泛用于许多研究,以确定调节性T细胞功能的肠道炎症。 仅将来自不同供体的原始T细胞或与分离自不同供体的Treg细胞一起转移至免疫缺陷型Rag1 -/- 小鼠中,并通过体重损失和评分分析评估疾病进展。 这种模型为肠炎症的研究提供了可靠的工作系统。

关键字:诱导的结肠炎, 小鼠模型, T细胞, 肠道炎症, RAG1 /—


  1. 15ml聚丙烯锥形管(Corning,目录号:430790)
  2. 50ml聚丙烯锥形管(Corning,目录号:430828)
  3. LS柱(Miltenyi Biotec,目录号:130-042-401)
  4. 用于供体(男性)的6-8周龄C57BL/6J WT小鼠
  5. 6-8周龄免疫缺陷型RAG1 -/- 小鼠(雄性)(Jackson Laboratory,股票号:002216)
  6. 10cm培养皿(Corning,目录号:430167)
  7. 10ml注射器(BD Biosciences,目录号:301604)
  8. 27号针(BD Biosciences,目录号:305109)
  9. 40μm尼龙细胞过滤器(Corning,Falcon ,目录号:352340)
  10. 70μm细胞过滤器(Corning,Falcon ,目录号:352350)
  11. 聚苯乙烯圆底流管(Corning,Falcon ,目录号:352054)
  12. FBS(Thermo Fisher Scientific,Gibco TM ,目录号:10100-147)
  13. RPMI培养基1640(Thermo Fisher Scientific,Gibco TM ,目录号:11875-093)
  14. ACK裂解缓冲液用于红细胞裂解(Thermo Fisher Scientific,Gibco TM ,目录号:A10492-1)
  15. 小鼠CD4阴性分离试剂盒(Miltenyi Biotec,目录号:130-095-248)
  16. 荧光标记抗体:
    1. 抗CD25-PE(BioLegend,目录号:102012)
    2. 抗CD4-PE-Cy7(BD Biosciences,目录号:563933)
    3. 抗CD44-APC(BD Biosciences,目录号:559250)
    4. 抗CD62L-FITC(BD Biosciences,目录号:553150)
  17. 37%甲醛(福尔马林)溶液(Avantor Performance Materials,J.T.Baker ,目录号:2016)
  18. FACS缓冲液(见配方)


  1. 普通玻璃显微镜载玻片(Omano,目录号:OMSK-50PL)
  2. 血细胞计数器
  3. 光学显微镜
  4. 顶部装入余额
  5. 离心机
  6. 流式细胞术(BD Biosciences,型号:ARIA III)


  1. 使用磁珠富集CD4 + T细胞
    1. 安乐死供体WT小鼠。
    2. 从鼠收获脾脏。
    3. 将解剖的器官在10厘米培养皿与10毫升PBS + 2%FBS。使用两片无菌磨砂玻璃(保持在-20°C提前1小时)载玻片轻轻研磨组织。有关详细的磨削程序,请参阅 http://www.bio-protocol。 org/e834
    4. 将70μm细胞过滤器置于50ml管的顶部,并将液体从盘转移至过滤器
    5. 在4℃,300×g下旋转脾悬液5分钟(注意:这应该是所有步骤的离心条件。)。通过抽吸弃去上清液,小心不要打扰药丸
    6. 加入4ml ACK裂解缓冲液,并在室温下孵育3分钟。再加入10ml PBS + 2%FBS
    7. 将40μm细胞过滤器置于50ml管的顶部,并将流体从50ml管转移至过滤器。旋转5分钟,倒掉上清液。
    8. 用10ml PBS + 2%FBS洗涤细胞2次。用PBS + 2%FBS缓冲液重悬细胞。取等分试样计数细胞。加入10微升的细胞悬浮液到90微升的台盼蓝溶液(1:10稀释),并使用血细胞计数器计数细胞。
    9. 通过使用miltenyi CD4 + T细胞分离试剂盒富集CD4 + T细胞。
    10. 按照阴性选择方案,通过在4℃下以300×g离心10分钟沉淀细胞。
    11. 通过抽吸弃去大部分上清液;通过小心吸取上下数次,将细胞沉淀重悬在1ml PBS + 2%FBS缓冲液中。取等分试样计数细胞。加入10微升的细胞悬浮液到90微升的台盼蓝溶液(1:10稀释),并使用血细胞计数器计数细胞。

  2. 细胞表面抗原染色用于细胞分选
    1. 我们将适当数量的细胞转移到FACS管中,并在300-400×g下旋转5分钟。
    2. 准备含有用含2%FBS的1×PBS稀释至最佳浓度(1:100)的抗CD4-PE-Cy7,抗CD25-PE,抗CD44-APC,抗CD62L-FITC抗体的抗体混合物。对于每一百万个细胞,我们通常添加100μl1×PBS与2%FBS缓冲液与稀释的抗体孵育。根据比率和要染色的细胞,制备抗体/PBS主混合物
    3. 重悬细胞在含有2%FBS的1×PBS中,以获得25-50×10 6个细胞/ml的浓度。细胞的浓度取决于待使用的特定细胞分选仪。将上述混合物加入细胞沉淀中。涡旋混合好。在4℃下染色15分钟。
    4. 通过用1x PBS + 2%FBS填充管来洗涤细胞两次。在4℃下以300×g离心5分钟。倒出液体。
    5. 如果可见碎屑或团块,在细胞分选之前需要使细胞通过40μm细胞过滤器
    6. 保护细胞免受光照,并在分选前置于冰上
    7. 将天然T细胞(CD4阳性+ CD25阳性CD44阳性CD62L阳性)和Treg群体(CD4 + CD25 hi )注射到含有2%FBS的RPMI培养基1640中。
  3. 用流动纯化的幼稚T细胞和Treg细胞注射RAG1 /- 小鼠
    1. 分选后,用冷的1x PBS填充含有分选的细胞的收集管,以如上所述通过离心收集壁中的细胞和沉淀。始终保持细胞在冰上,以避免在每一步中聚集
    2. 用冷的1x PBS洗涤细胞2次。此步骤旨在耗尽跟踪FBS。非常轻轻地重悬细胞,并准备与PBS的单细胞悬浮液,并在注射前将细胞置于冰上。取等分试样计数细胞。添加10微升的细胞悬液到90微升的台盼蓝溶液(1:10稀释),并取10微升稀释的细胞悬液,使用血细胞计数器计数细胞。
    3. 计数后,用冷的1x PBS稀释细胞,并调节至2×10 6/ml的最终细胞浓度。在注射之前将注射管保持在冰上。
    4. 混合不同组的细胞。对于每只小鼠,将4×10 5个天然T细胞单独(200μl)或4×10 5个初始T(200μl)连同2×10 5个5 Treg细胞(100μl)混合
    5. 称重受体小鼠使用顶部负载平衡和记录重量。在注射小鼠之前,轻轻地倒转管以混合细胞。
    6. 限制鼠标使用尾部访问鼠标限制器。有关详细限制器图片,请参阅: http://www .stoeltingco.com/neuroscience/misc/restrainers.html 。稍微旋转尾部以显现静脉。非常缓慢地将细胞吸入1毫升注射器与27号针连接。将针头轻轻插入静脉,注射所有细胞。

  4. 监测疾病进展和结肠炎评估
    注意:一些小鼠不会出现明显的大便和腹泻。然而,这些小鼠仍然可能发展显着的肠炎症,这将在H& E染色时可见。
    1. 当小鼠失去其原始体重的约15-20%或在注射后约40天时,应对动物实施安乐死并评估结肠炎的证据。
    2. 从肛门小心切除整个结肠,切除它的肠系膜从盲肠下面到肛门开口上方。小心不要伸展结肠。
    3. 将结肠放在塑料板上,测量并记录其长度。然后通过注射PBS清洁粪便。称重结肠组织
    4. 可以记录结肠长度和重量以计算长度重量比,其已经显示与盲的组织病理学评分良好相关。
    5. 对于组织学加工和盲目的组织病理学评分,获得从结肠的近端和远端部分的小(0.5cm)片。然后将这些组织片置于10%PBS-福尔马林中,并在石蜡包埋之前在室温下固定至少24小时。
    6. 将固定组织嵌入石蜡块。石蜡包埋样品的截面产生4μm切片。在微观载玻片上安装三个切片,用苏木精和曙红染色切片
    7. 使用以下评分标准进行盲法组织病理学分析:
      1. 固有层炎症程度(0-3分)
      2. 杯状细胞丢失(得分0-2)
      3. 异常隐窝(得分0-3)
      4. 存在隐窝脓肿(得分0-1)
      5. 粘膜侵蚀和溃疡(0-3分)
      6. 黏膜下粘膜扩散至透壁受累(分值0-3)
      7. 以40×放大倍数计数的嗜中性粒细胞数(分数0-4)


图1.初始T细胞标记物的示意图。将脾细胞重悬并用含有抗CD4-PE-Cy7,抗CD25-PE,抗CD44-APC,抗CD44- CD62L-FITC抗体在4℃孵育15分钟。如上所述门控天然T细胞(CD4 + CD25 + CD44 + CD62L hi )。

图2:如何研究结肠炎的T细胞转移模型的示意图。每个Rag1 受体小鼠用4×10 5个/sup /分离自野生型小鼠的CD4 + CD25L初级T细胞和CD4 + CD25 +初级CD44L + CD4 + CD25L初级T细胞,用从WT分离的2×10 5个CD4 + Treg细胞(b)转移。(a)中的小鼠的体重减轻是从注射后第40天开始每四天监测一次。每组6只小鼠。 (c)来自未处理的WT小鼠(对照),用WT天然T细胞(天然),Rag1 -/- 小鼠转移的Rag1 -/- 小鼠的结肠切片的代表性H&与初始T细胞和WT Treg细胞(初始+ WT Treg(原始放大倍数,20x))共转染的小鼠。


  1. 转移导致没有结肠炎发展。可能的原因:
    1. 转移低天然T细胞数。
      细胞活力是另一个关键因素。确保细胞总是保持在冰上。重要的是在分选完成后评估细胞活力,并且只有> 90%的生存力是可接受的转移。
  2. 重要的是要记住,这种模型取决于许多额外的因素,除了幼稚T细胞,充足的幼稚T细胞和良好的动物程序的可行性。这些包括动物房屋的清洁和受体小鼠中完整的肠道菌群。


  1. FACS缓冲区


这个协议是从我们以前的工作Pan 等人修改(2015)。这项工作得到了973项目(2014CB541902,2014CB541901),中国国家自然科学基金(81230072,81025016,31370880),中国科学院重点研究计划(KSZD-EW-Z-003-3) ,以及中国卫生部(201202008)和上海科学技术委员会计划(12431900703,12JC1406000,12ZR1435900)。


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引用:Pan, W., Zhu, S., Dai, D., Tang, Y., Yao, Y. and Shen, N. (2016). T Cell Transfer Model of Colitis. Bio-protocol 6(13): e1862. DOI: 10.21769/BioProtoc.1862.