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Imaging Cytokine Concentration Fields Using PlaneView Imaging Devices
使用PlaneView成像装置对细胞因子浓度视野进行成像   

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Immunity
Apr 2017

 

Abstract

We describe here a method to visualize concentration fields of cytokines around cytokine-secreting cells. The main challenge is that physiological cytokine concentrations can be very low, in the pico-molar range. Since it is currently impossible to measure such concentrations directly, we rely on cell’s response to the cytokines–the phosphorylation of a transcription factor–that can be visualized through antibody staining. Our devices aim at mimicking conditions in dense tissues, such as lymph nodes. A small number of secreting cells is deposited on a polylysine-coated glass and covered by multiple layers of cytokine-consuming. The cells are left to communicate for 1 h, after which the top layers are removed and the bottom layer of cells is antibody labeled for the response to cytokines. Then a cross-section of cytokine fields can be visualized by standard fluorescence microscopy. This manuscript summarized our method to quantify the extent of cytokine-mediated cell-to-cell communications in dense collection of cells in vitro.

Keywords: Cytokine concentration (细胞因子浓度), Cytokine niches (细胞因子微环境), Imaging of cytokine fields (细胞因子视野成像)

Background

The mammalian immune system has evolved to identify and limit the spread of potential pathogens while minimizing collateral tissue damage caused by the immune system itself. To achieve this, immune cells rely on a network of cytokine mediators that enable cell-to-cell communications and broadcast information about the magnitude and nature of the pathogenic insult. Vast arrays of different cytokines bind strongly to their cognate receptors, often with characteristic binding affinities in the nano- or pico-molar range. Immunological niches are generated via cytokine communications. For example, in both the bone marrow and the thymus, secretion of Interleukin-7 (IL-7) by stromal cells supports the survival of proliferating B and T cell progenitors, respectively (Tokoyoda et al., 2004; Alves et al., 2009). The size of the cytokine niche controls the number of maturing progenitors, thereby keeping the blood cell compartments in equilibrium (Böyum, 1968; Weist et al., 2015).

We aim to collect information about the spatial and temporal dynamics of cytokines and how these two parameters influence the immune response. This is an area of immunology that is currently under-studied. Many assays test the effects of cytokines in tissue-culture dishes, where media is well-mixed, leading to homogeneous fields of growth and differentiation factors. The intricate and highly specialized architecture of the secondary lymphoid organs sets up niches where cells sense stimuli such as pathogen components and cytokines, proliferate, mature, differentiate, and die. Cytokine concentration gradients are formed within these niches such that some cells have greater or lesser access to cytokines than others (Liu et al., 2015). Measuring how far cytokines spread from their source, and the gradients they form, is key to unravelling the mechanism of the phenotypic heterogeneity of immune cells in differentiation, proliferation, and death (Feinerman et al., 2010; Busse et al., 2010; Höfer et al., 2012; Müller et al., 2012; Thurley et al., 2015).

Due to the typically low concentrations (pM range) of free cytokines in vivo, direct measurement of cytokine fields is difficult at best and maybe impossible. However, due to their high sensitivity to cytokine and graded, concentration-dependent response, the signaling levels of cells in response to cytokines can itself be used as a bio-sensor for cytokine concentrations (Oyler-Yaniv et al., 2017).

In this protocol, we describe how to directly image the signaling response generated around a cytokine producer in vitro, in conditions that mimic in vivo conditions: high cell density and no convection. Our method is general and can be applied to any cell type and any diffusible stimulus, and only depends on the existence of a specific antibody to target the downstream signaling molecule of interest and/or of live cell reporters.

Materials and Reagents

  1. Pipette tips (USA Scientific, catalog numbers: 1111-1806 , 1111-3800 )
  2. CELLSTAR Filter Cap Cell Culture Flasks T75 (Greiner Bio One International, catalog number: 658175 )
  3. 15 ml tube
  4. Glass slides (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4951PLUS4 )
  5. Coverslips (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 25X25-1 )
  6. Silicone rubber compound (PDMS) (Momentive, catalog number: RTV615 )
  7. PVP-treated PCTE Membranes, 13 mm diameter, 400 nm pore (Sterlitech, catalog number: PCT0413100 )
  8. B16-F10 melanoma cells (ATCC, catalog number: CRL-6475 )
  9. Mouse CD4 (L3T4) MicroBeads (Miltenyi Biotec, catalog number: 130-049-201 )
  10. Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, catalog number: P1585-1MG )
  11. Ionomycin calcium salt (Sigma-Aldrich, catalog number: I0634-1MG )
  12. Ficoll-paque plus (GE Healthcare, catalog number: 17144003 )
  13. Recombinant mouse IL-2 (Thermo Fisher Scientific, eBioscienceTM, catalog number: 14-8021-64 )
  14. Recombinant human IL-2 (gift from Dr. Kendall A. Smith, Cornell University)
  15. Trypsin/EDTA solution (Thermo Fisher Scientific, GibcoTM, catalog number: R001100 )
  16. Phosphate buffered saline (Sigma-Aldrich, catalog number: P4417 )
  17. Glycine (Sigma-Aldrich, catalog number: 50046 )
  18. Ovalbumin peptide SIINFEKL (Sigma-Aldrich, catalog number: S7951-1MG )
  19. Cell Trace Far-Red (DDAO-SE) (Thermo Fisher Scientific, InvitrogenTM, catalog number: C34564 )
  20. Poly-L-lysine solution (Sigma-Aldrich, catalog number: P8920-100ML )
  21. Paraformaldehyde solution, 4% in PBS (Alfa Aesar, Affymetrix, catalog number: J19943 )
  22. Methanol (Sigma-Aldrich, catalog number: MX0490-4 )
  23. Fluoromount Aqueous Mounting Medium (Sigma-Aldrich, catalog number: F4680-25ML )
  24. Triton 100-X (MP Biomedicals, catalog number: 0230022101-1l )
  25. α-CD4, Alexa700, Pacific Blue (BD Bioscience clone RM4-5, BD, catalog numbers: 557956 , 558107 )
  26. α-IL-2Rα, PE (Miltenyi Biotec clone 7D4, Miltenyi Biotec, catalog number: 130-102-593 )
  27. Primary antibody rabbit α-phospho-STAT5 (pY694) (Cell Signaling clone C71E5, Cell Signaling Technology, catalog number: 9314S )
  28. Primary antibody rabbit α-phospho-STAT1 (pY701) (Cell Signaling clone 58D6, Cell Signaling Technology, catalog numbers: 9167L )
  29. Secondary polyclonal antibody α-rabbit IgG, Alexa 488 (Jackson ImmunoResearch, catalog number: 711-176-152 )
  30. RPMI 1640 media with L-glutamine (Biological Industries, catalog number: 01-100-1A )
  31. Heat-inactivated fetal bovine serum (Biological Industries, catalog number: 04-127-1A )
  32. HEPES buffer (Biological Industries, catalog number: 03-025-1B )
  33. Non-essential amino acids (Biological Industries, catalog number: 01-340-1B )
  34. Sodium pyruvate (Biological Industries, catalog number: 03-042-1B )
  35. Penicillin-streptomycin solution (Biological Industries, catalog number: 03-031-5B )
  36. β-Mercaptoethanol (Sigma-Aldrich, catalog number: M3148-25ML )
  37. Complete RPMI (see Recipes)

Equipment

  1. Pipettes
  2. Heraeus centrifuge with microplate swinging rotor (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM BiofugeTM StratosTM )
  3. Zeiss Axiovert 200M microscope (ZEISS, model: Axiovert 200M )

Software

  1. MATLAB, Mathworks Inc.
  2. LabVIEW, National Instruments

Procedure

The protocols described here were developed for imaging of cytokine signaling in dense clusters of cytokine consuming cells that are simulated in vivo conditions in secondary lymphoid organs or other solid tissue (Oyler-Yaniv et al., 2017).

  1. Cell culture
    1. OT-I and C57BL/6 primary cells are harvested from the lymph nodes and spleen, and mechanically separated into single-cell suspension. CD4+ T-cells are isolated using Mouse CD4 (L3T4) MicroBeads. Primary cells and B16-F10 melanoma cells (ATCC CRL-6475) are maintained in complete RPMI (Recipe 1) throughout the procedure. C57BL/6 T cells are activated using 10 ng/ml PMA and 500 ng/ml Ionomycin and cultured in 30 ml of media, in a T75 flask for 3 days.
    2. Dead cells are removed using Ficoll-paque plus density gradient media following standard protocols (Böyum, 1968) and subsequently cultured at 106 cells/ml in RPMI supplemented with 2 nM recombinant human IL-2 for an additional day. IL-2 secretion experiments are performed on day 4 of culture.
    3. B16-F10 cells are maintained in 30 ml of complete RPMI (Recipe 1) in a T75 flask and passaged every 3 days using trypsin/EDTA solution and recultured to 10% confluency (roughly 106 cells at the start of each passage). Cells are never used beyond passage 7.
      Note: B16-F10 melanoma cells express the IFNγ receptor but do not produce IFNγ. They also express high levels of MHC-I and therefore serve as antigen presenting cells for TCR recognition.

  2. Preparation of cells for imaging
    1. To remove receptor-bound cytokines, wash 5 x 107 cultured T-cells (consumer cells) in 10 ml PBS, then expose 0.1 M glycine in PBS (pH 4.0) for 1 min on ice, followed by 3 washes each with 10 ml RPMI, and rest for 1 h at in a tube containing RPMI in a 37 °C incubator.
    2. To generate CD4 depleted, naïve splenocytes (inert cells), harvest lymph nodes and spleen from a C57BL/6 mouse. Separate tissue into single-cell suspension. Deplete the cell suspension of CD4+ cells using Mouse CD4 (L3T4) MicroBeads.
    3. Mix cultured consumer cells with inert cells at different ratios.
    4. Re-administrate the previously activated T-cells cultures with 5 ng/ml PMA and 500 ng/ml Ionomycin to generate IL-2 producing cells. This reactivation leads to a rapid and extensive production of IL-2 by the T-cells. These cells are labeled with DDAO-SE by using the manufacturer’s protocol at 1 µM for subsequent identification. Only the cytokine producers are labeled at this stage!
    5. Pulse 107 B16-F10 cells with 1 nM of the SIINFEKL peptide in 10 ml of RPMI for 1 h under constant rotation in a 15 ml tube, in an incubator. OT-I Cells are labeled with DDAO-SE by using the manufacturer’s protocol at 1 µM for subsequent identification.

  3. General description: Fabrication of imaging devices
    1. Coat glass slides with poly-L-lysine (PLL) by submerging them in 0.01% PLL, diluted in H2O, for 40 min at 37 °C. Wash slides by submerging them in a large volume of H2O and allow them to dry at room temperature for 1 h. Place a small (6 mm) hollow cylinder made of PDMS on the slide, the PDMS rapidly attaches to the slide creating a small well.
    2. To create a tightly packed cell pellet, cell suspensions are added into the PDMS well, the slide is put inside a pipette box cover and centrifuged for 1 min at 800 x g to allow cells to stick to the glass slide (Figure 1A). As more cells accumulate on the device, they form a 3 dimensional layered structure. Depending on cell dimensions, each layer will contain ~1-2 x 105 individual cells. Moreover, different layers composed of different cell preparations can be added sequentially, creating a stratified structure that can mimic specific in vivo morphologies.
    3. After cell deposition, the PDMS well is removed and a 13 mm diameter semipermeable hydrophilic membrane is dipped in media and carefully placed on top of the cells. This membrane protects the cells from moving due to changing reagents, and prevents convection flows from distorting the concentration fields. The cells and membrane are then covered by a larger PDMS well (12 mm diameter) to allow for the confinement of reagents around the cells. At the end of the experiment, the cells are fixed for 20 min in 200 μl 37 °C 4% PFA and permeabilized by using 200 μl ice cold 90% MeOH for 10 min. After incubation and fixation, cells that are in contact with the glass slide remain permanently bound to it, preserving their spatial distribution. The PDMS well and the membrane are then removed using forceps, the slides are stained using standard immunofluorescence protocols, and finally are mounted on a coverslip using Fluoromount (Figure 1B). The process is illustrated in Figure 2.


      Figure 1. Preparation of slides for imaging. A. PlaneView device after centrifugation; B. Cell patch after staining and mounting.


      Figure 2. Graphical protocol of device preparation. Preparation of cells for imaging is done by first coating a glass slide with poly-L-lysine (1). Then, a cell suspension containing a small fraction of cytokine producing cells is deposited in a monolayer by centrifugation (2, 3). 10 layers of cells containing no producers is deposited on top to form a 3 dimensional structure (4, 5). The cells are covered with a semipermeable membrane (6), incubated for 1 h (7), and fixed in situ (8). Cells are then permeabilized and stained (9, 10).

  4. In vitro imaging of cytokine concentration fields
    1. For measuring IL-2 concentration fields (Figure 3), 2 x 105 IL-2 consuming T cells, or a combination of 10% consuming T cells (2 x 104) and 90% inert cells (1.8 x 105) are mixed with 0.1% IL-2 producing T cells (200 cells), each in a total volume of 20 μl, and deposited in a monolayer. Then, 10 more layers (2 x 106) of cells containing no producers and either a 1:0 or a 1:9 ratio of consumers to inert cells, respectfully, each in a volume of 180 μl, are added on top, forming a three dimensional strata with the producing cells dispersed on the bottom (Figure 2). A semipermeable membrane is placed on the cells to preserve their positions during further processing.


      Figure 3. pSTAT5 distribution around IL-2 producers. Immunofluorescence staining of cell preparations containing either 100% IL-2Rα+ consuming cells or 10% consuming cells and 90% IL-2Rα- inert cells, and a small number (< 0.01%) of IL-2 producing T cells in a PlaneView imaging device. Blue: DAPI, Red: IL-2Rα, Green: pSTAT5, Magenta: DDAO-SE.

    2. For measuring IFNγ concentration fields (Figure 4), 105 SIINFEKL pulsed B16-F10 melanoma cells are mixed with 0.2% OT-I T cells (200 cells) and deposited in a monolayer. Then, 10 more layers of B16-F10 cells (106) are added on top. A semipermeable membrane is placed on the cells to preserve their positions during further processing.


      Figure 4. pSTAT1 distribution around IFNγ producer. Immunofluorescence staining of cell preparation containing SIINFEKL pulsed B16-10A cells spiked with a small amount of OT-I cells was prepared as described in Procedure D. Blue: DAPI, Red: pSTAT1, Green: DDAO-SE.

    3. To control for background transcription factor phosphorylation and cytokine specificity, devices are loaded with consuming cell cultures containing no producers. The cells are then covered with either fresh media or media containing 10 nM of cytokine to serve as negative and positive controls, respectively. These samples showed effectively no signal for the negative control and bright, uniform, signal for the positive control (Oyler-Yaniv et al., 2017).
    4. The system is incubated at 37 °C for 1 h. After that, media is carefully aspirated off the devices and the cells are fixed for 20 min at 37 °C in 4% PFA. PFA is then removed and the cells are permeabilized by using 200 μl ice cold 90% MeOH for 10 min to allow for intracellular immunostaining. Special attention should be given during this stage to minimally disrupt the cell pellets.
    5. After fixation and permeabilization, the PDMS well and the membrane are removed using forceps. At this stage, the cells would be tightly bound to the glass slide and standard immunostaining protocols can be used. Nonspecific antibody binding is blocked by a 1 h incubation in 5% FBS and 0.3% Triton X-100 in PBS at room temperature. Primary antibodies (Rabbit anti-pSTAT5 1:200) are applied in a moist chamber for 1 h at room temperature. Fluorophore-conjugated antibodies (Goat anti-Rabbit Alexa 488, Rat anti-IL-2Rα R-PE, 1:300) are applied for 1 h at room temperature. Cells are then briefly stained with DAPI and a coverslip is mounted using Fluoromount (Figure 1B) for fluorescent imaging.

Data analysis

Images are processed by segmenting individual cells (Gonzalez and Woods, 2007), then determining whether each cell is a consumer based on IL-2Rα expression. On each consumer, the total level of pSTAT5 expression and the center-of-mass is calculated and logged. An example of the procedure is shown in Figure 5. Profiles of pSTAT5 expression on individual cells as a function of distance from the nearest producer are then generated (Figure 6).


Figure 5. Image processing procedure


Figure 6. Analysis of pSTAT5 distributions. A. A sample containing 100% IL-2 consuming T cells spiked with a small amount of IL-2 producing cells was prepared as described in Procedure D. B. Image reconstruction based on total pSTAT5 and center of mass per cell. The radius of each circle is proportional to the pSTAT5 level. C. pSTAT5 profile as a function of the distance from the cytokine producer.

Recipes

  1. Complete RPMI
    RPMI 1640 media supplemented with:
    Heat-inactivated 10% fetal bovine serum
    2 mM L-glutamine
    10 mM HEPES
    0.1 mM non-essential amino acids
    1 mM sodium pyruvate
    100 µg/ml of penicillin
    100 µg/ml of streptomycin
    50 µM β-mercaptoethanol

Acknowledgments

This work has been done in collaboration with Grégoire Altan-Bonnet (NIH) whom we would like to thank for the overall support and, in particular, for the critical reading of the manuscript. We are also grateful to U.S.-Israel Binational Science Foundation (grant #2012327 to G. Altan-Bonnet and O.K.) for funding. The protocol has been adapted from Oyler-Yaniv et al., 2017.
The Authors declare no conflicts of interest or competing interests.

References

  1. Alves, N. L., Richard-Le Goff, O., Huntington, N. D., Sousa, A. P., Ribeiro, V. S., Bordack, A., Vives, F. L., Peduto, L., Chidgey, A., Cumano, A., Boyd, R., Eberl, G. and Di Santo, J. P. (2009). Characterization of the thymic IL-7 niche in vivo. Proc Natl Acad Sci U S A 106(5): 1512-1517.
  2. Böyum, A. (1968). Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl 97: 77-89.
  3. Busse, D., de la Rosa, M., Hobiger, K., Thurley, K., Flossdorf, M., Scheffold, A. and Hofer, T. (2010). Competing feedback loops shape IL-2 signaling between helper and regulatory T lymphocytes in cellular microenvironments. Proc Natl Acad Sci U S A 107(7): 3058-3063.
  4. Feinerman, O., Jentsch, G., Tkach, K. E., Coward, J. W., Hathorn, M. M., Sneddon, M. W., Emonet, T., Smith, K. A. and Altan-Bonnet, G. (2010). Single-cell quantification of IL-2 response by effector and regulatory T cells reveals critical plasticity in immune response. Mol Syst Biol 6: 437.
  5. Gonzalez, R. C. and Woods, R. E. (2007). Digital Image Processing (3rd Edition).
  6. Höfer, T. O. Krichevsky, O, and Altan-Bonnet, G. (2012). Competition for IL-2 between regulatory and effector T cells to chisel immune responses. Front Immunol 3: 268.
  7. Liu, Z., Gerner, M. Y., Van Panhuys, N., Levine, A. G., Rudensky, A. Y. and Germain, R. N. (2015). Immune homeostasis enforced by co-localized effector and regulatory T cells. Nature 528(7581): 225-230.
  8. Müller, A. J., Filipe-Santos, O., Eberl, G., Aebischer, T., Spath, G. F. and Bousso, P. (2012). CD4+ T cells rely on a cytokine gradient to control intracellular pathogens beyond sites of antigen presentation. Immunity 37(1): 147-157.
  9. Oyler-Yaniv, A., Oyler-Yaniv, J., Whitlock, B. M., Liu, Z., Germain, R. N., Huse, M., Altan-Bonnet, G. and Krichevsky, O. (2017). A tunable diffusion-consumption mechanism of cytokine propagation enables plasticity in cell-to-cell communication in the immune system. Immunity 46(4): 609-620.
  10. Thurley, K., Gerecht, D., Friedmann, E. and Hofer, T. (2015). Three-dimensional gradients of cytokine signaling between T cells. PLoS Comput Biol 11(4): e1004206.
  11. Tokoyoda, K., Egawa, T., Sugiyama, T., Choi, B. I. and Nagasawa, T. (2004). Cellular niches controlling B lymphocyte behavior within bone marrow during development. Immunity 20(6): 707-718.
  12. Weist, B. M., Kurd, N., Boussier, J., Chan, S. W. and Robey, E. A. (2015). Thymic regulatory T cell niche size is dictated by limiting IL-2 from antigen-bearing dendritic cells and feedback competition. Nat Immunol 16(6): 635-641.

简介

我们在这里描述了一种可视化细胞因子分泌细胞周围细胞因子浓度场的方法。主要挑战是生理细胞因子浓度可能非常低,在微摩尔浓度范围内。由于目前不可能直接测量这样的浓度,我们依赖于细胞对细胞因子的反应 - 转录因子的磷酸化 - 可以通过抗体染色显现。我们的设备旨在模仿密集组织中的条件,如淋巴结。少数分泌细胞沉积在聚赖氨酸包被的玻璃上并被多层细胞因子消耗覆盖。将细胞连通1小时,之后去除顶层,并且细胞的底层被抗体标记为对细胞因子的应答。然后通过标准荧光显微镜观察细胞因子场的横截面。这篇手稿总结了我们的方法,以量化密集细胞体外细胞因子介导的细胞间通讯的程度。

【背景】哺乳动物的免疫系统已经发展到能够识别和限制潜在病原体的传播,同时使由免疫系统本身造成的附带组织损伤最小化。为了实现这一点,免疫细胞依赖细胞因子介质网络,这些细胞因子介质能够进行细胞间通讯并广播关于致病性侮辱的大小和性质的信息。大量不同细胞因子与其同源受体强烈结合,通常在纳摩尔或皮摩尔范围内具有特征性结合亲和力。通过细胞因子通讯产生免疫龛。例如,在骨髓和胸腺中,通过基质细胞分泌的白细胞介素-7(IL-7)分别支持增殖的B细胞和T细胞祖细胞的存活(Tokoyoda et al。, 2004; Alves等人,2009)。细胞因子生态位的大小控制成熟祖细胞的数量,从而保持血细胞区室平衡(Böyum,1968; Weist等人,2015)。

我们旨在收集有关细胞因子的空间和时间动态以及这两个参数如何影响免疫反应的信息。这是一个目前正在研究中的免疫学领域。许多分析测试细胞因子在组织培养皿中的效果,其中培养基充分混合,导致均质的生长和分化因子领域。次级淋巴器官的错综复杂和高度专业化的结构为细胞感受刺激如病原体成分​​和细胞因子,增殖,成熟,分化和死亡奠定了基础。细胞因子浓度梯度在这些龛中形成,使得一些细胞比其他细胞有更多或更少的细胞因子(Liu等人,2015)。测量细胞因子从其来源扩散的程度以及它们形成的梯度是揭示免疫细胞在分化,增殖和死亡中的表型异质性的机制的关键(Feinerman et al。,2010; Busse等人,2010;Höfer等人,2012;Müller等人,2012; Thurley等人。,2015)。

由于体内游离细胞因子的浓度(pM范围)通常较低,所以细胞因子场的直接测量很难,也许是不可能的。然而,由于它们对细胞因子的高度敏感性和分级的浓度依赖性响应,细胞对细胞因子的响应信号水平本身可以用作细胞因子浓度的生物传感器(Oyler-Yaniv等人 ,2017)。

在这个协议中,我们描述了如何在模拟体内条件下直接成像细胞因子生产者周围产生的信号传导应答:高细胞密度和不对流。我们的方法是通用的,可应用于任何细胞类型和任何扩散性刺激,并且仅取决于特定抗体的存在以靶向感兴趣的下游信号分子和/或活细胞报道分子。

关键字:细胞因子浓度, 细胞因子微环境, 细胞因子视野成像

材料和试剂

  1. 移液器吸头(USA Scientific,产品目录号:1111-1806,1111-3800)
  2. CELLSTAR过滤瓶盖细胞培养瓶T75(Greiner Bio One International,目录号:658175)
  3. 15毫升管
  4. 玻璃载玻片(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:4951PLUS4)
  5. 盖帽(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:25X25-1)
  6. 硅橡胶化合物(PDMS)(Momentive,目录号:RTV615)
  7. PVP处理的PCTE膜,13mm直径,400nm孔(Sterlitech,目录号:PCT0413100)
  8. B16-F10黑素瘤细胞(ATCC,目录号:CRL-6475)
  9. 小鼠CD4(L3T4)微珠(Miltenyi Biotec,目录号:130-049-201)
  10. 佛波醇12-肉豆蔻酸酯13-乙酸酯(PMA)(Sigma-Aldrich,目录号:P1585-1MG)
  11. 离子霉素钙盐(Sigma-Aldrich,目录号:I0634-1MG)
  12. Ficoll-paque plus(GE Healthcare,目录号:17144003)
  13. 重组小鼠IL-2(Thermo Fisher Scientific,eBioscience TM,产品目录号:14-8021-64)
  14. 重组人IL-2(来自康奈尔大学Kendall A. Smith博士的礼物)
  15. 胰蛋白酶/ EDTA溶液(Thermo Fisher Scientific,Gibco TM,目录号:R001100)
  16. 磷酸盐缓冲盐水(Sigma-Aldrich,目录号:P4417)
  17. 甘氨酸(Sigma-Aldrich,目录号:50046)
  18. 卵清蛋白肽SIINFEKL(Sigma-Aldrich,目录号:S7951-1MG)
  19. 细胞追踪远红(DDAO-SE)(Thermo Fisher Scientific,Invitrogen TM,目录号:C34564)
  20. 聚-L-赖氨酸溶液(Sigma-Aldrich,目录号:P8920-100ML)
  21. 多聚甲醛溶液,4%于PBS中(Alfa Aesar,Affymetrix,目录号:J19943)
  22. 甲醇(Sigma-Aldrich,目录号:MX0490-4)
  23. Fluoromount水性固定介质(Sigma-Aldrich,目录号:F4680-25ML)
  24. Triton 100-X(MP Biomedicals,目录号:0230022101-1)
  25. α-CD4,Alexa700,Pacific Blue(BD Bioscience克隆RM4-5,BD,目录号:557956,558107)
  26. α-IL-2Rα,PE(Miltenyi Biotec clone 7D4,Miltenyi Biotec,目录号:130-102-593)
  27. 第一抗体兔α-磷酸-STAT5(pY694)(Cell Signaling clone C71E5,Cell Signaling Technology,目录号:9314S)
  28. 第一抗体兔α-磷酸-STAT1(pY701)(Cell Signaling clone 58D6,Cell Signaling Technology,目录号:9167L)
  29. 二级多克隆抗体α-兔IgG,Alexa 488(Jackson ImmunoResearch,目录号:711-176-152)
  30. 含L-谷氨酰胺的RPMI 1640培养基(Biological Industries,目录号:01-100-1A)
  31. 热灭活的胎牛血清(Biological Industries,目录号:04-127-1A)
  32. HEPES缓冲液(Biological Industries,目录号:03-025-1B)
  33. 非必需氨基酸(Biological Industries,目录号:01-340-1B)
  34. 丙酮酸钠(Biological Industries,目录号:03-042-1B)
  35. 青霉素 - 链霉素溶液(Biological Industries,目录号:03-031-5B)
  36. β-巯基乙醇(Sigma-Aldrich,目录号:M3148-25ML)
  37. 完整的RPMI(见食谱)

设备

  1. 移液器
  2. 带有微板摇摆转子的Heraeus离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus TM Biofuge TM Stratos TM TM >)
  3. 蔡司Axiovert 200M显微镜(蔡司,型号:Axiovert 200M)

软件

  1. MATLAB,Mathworks公司
  2. LabVIEW,National Instruments

程序

这里描述的方案被开发用于在次级淋巴器官或其他实体组织中模拟体内条件的致密细胞因子消耗细胞簇中成像细胞因子信号传导(Oyler-Yaniv等人, / em>,2017)。

  1. 细胞培养
    1. 从淋巴结和脾收获OT-I和C57BL / 6原代细胞,并机械分离成单细胞悬液。使用小鼠CD4(L3T4)微珠分离CD4 + T细胞。在整个过程中将原代细胞和B16-F10黑素瘤细胞(ATCC CRL-6475)维持在完全RPMI(配方1)中。使用10ng / ml PMA和500ng / ml离子霉素活化C57BL / 6T细胞并在30ml培养基中在T75烧瓶中培养3天。
    2. 根据标准方案(Böyum,1968),使用Ficoll-paque加密度梯度培养基去除死细胞,随后在补充有2nM重组人IL-2的RPMI中以10 6细胞/ ml培养另外的细胞天。 IL-2分泌实验在培养的第4天进行。
    3. 将B16-F10细胞维持在Tml瓶中的30ml完全RPMI(配方1)中,并使用胰蛋白酶/ EDTA溶液每3天进行传代,并重新培养至10%融合(约10 6细胞每一段的开始)。细胞从来没有用过第7代。
      注:B16-F10黑素瘤细胞表达IFNγ受体,但不产生IFNγ。它们也表达高水平的MHC-I,因此可作为TCR识别的抗原呈递细胞。

  2. 制备用于成像的细胞
    1. 为了除去受体结合的细胞因子,在10ml PBS中洗涤5×10 7个培养的T细胞(消费者细胞),然后在PBS(pH 4.0)中将0.1M甘氨酸在冰上暴露1分钟,随后每次用10ml RPMI洗涤3次,并在含有RPMI的管中在37℃培养箱中静置1小时。
    2. 为了产生CD4耗尽的幼稚脾细胞(惰性细胞),从C57BL / 6小鼠收获淋巴结和脾脏。将组织分离成单细胞悬液。使用小鼠CD4(L3T4)微珠消耗CD4 + / +细胞的细胞悬液。
    3. 将培养的消费者细胞与不同比例的惰性细胞混合。
    4. 用5ng / ml PMA和500ng / ml伊屋诺霉素重新施用先前活化的T细胞培养物以产生产生IL-2的细胞。这种再激活导致T细胞迅速而广泛地产生IL-2。这些细胞用DDAO-SE通过使用1μM的制造商的方案进行标记以用于随后的鉴定。在这个阶段只有细胞因子生产者被标记!
    5. 在培养箱中在10ml RPMI中用10nM的SIINFEKL肽脉冲10 7 B16-F10细胞1小时恒定旋转。
      使用1μM制造商的协议,用DDAO-SE标记OT-I细胞用于后续识别

  3. 一般描述:成像设备的制造
    1. 通过将聚-L-赖氨酸(PLL)涂覆在含0.01%PLL的稀释于37℃的H 2 O中40分钟,使载玻片与聚-L-赖氨酸(PLL)一起滑动。通过将载玻片浸入大量H 2 O中清洗载玻片并使其在室温下干燥1小时。将一个由PDMS制成的小型(6毫米)中空圆柱体放置在载玻片上,PDMS快速连接到载玻片上,形成一个小孔。
    2. 为了产生紧密包装的细胞沉淀,将细胞悬浮液加入到PDMS孔中,将该载玻片置于移液管盒盖内并以800×g离心1分钟以使细胞粘附到载玻片上(图1A)。随着更多细胞在装置上累积,它们形成三维分层结构。根据细胞的大小,每层将包含〜1-2×10 5个单独的细胞。此外,由不同细胞制剂组成的不同层可以依次添加,形成可以模拟特定体内形态的分层结构。
    3. 细胞沉积后,去除PDMS孔,并将13mm直径的半透性亲水膜浸入培养基中并小心放置在细胞顶部。该膜可防止因试剂更换而引起的细胞移动,并防止对流流动造成浓度场扭曲。然后将细胞和膜用更大的PDMS孔(12mm直径)覆盖以允许细胞周围的试剂限制。在实验结束时,将细胞在200μl37℃4%PFA中固定20分钟,并通过使用200μl冰冷的90%MeOH透化10分钟。孵育和固定后,与载玻片接触的细胞将永久地与其保持结合,从而保持其空间分布。然后使用镊子将PDMS孔和膜去除,使用标准免疫荧光方案将载玻片染色,最后使用Fluoromount将其安装在盖玻片上(图1B)。该过程如图2所示。


      图1.制备用于成像的载玻片。 :一种。离心后的PlaneView装置; B.染色和安装后的细胞贴剂。


      图2.设备准备的图形协议。用于成像的细胞的制备通过首先用聚-L-赖氨酸(1)涂布载玻片来完成。然后,通过离心将含有少量细胞因子生成细胞的细胞悬液沉积在单层中(2,3)。 10层不含生产者的细胞沉积在顶部以形成三维结构(4,5)。用半透膜(6)覆盖细胞,孵育1小时(7),然后原位固定(8)。然后将细胞透化并染色(9,10)。

  4. 细胞因子浓度场的体外成像
    1. 为了测量IL-2浓度场(图3),2×10 5 IL-2消耗性T细胞或10%消耗性T细胞的组合(2×10 4 4 / sup )和90%惰性细胞(1.8×10 5)与0.1%产生IL-2的T细胞(200个细胞)混合,各自总体积为20μl,并沉积在单层。然后,分别将10个不含生产者的细胞层(2×10 6个细胞)和1:0或1:9的消耗者与惰性细胞的比率分别为180μl ,加在顶部,形成三维地层,生产细胞分散在底部(图2)。一个半透膜被放置在细胞上,以在进一步加工过程中保持它们的位置。


      图3. IL-2生产者周围的pSTAT5分布。含有100%IL-2Rα+消耗细胞或10%消耗细胞和90%IL-2Rα-惰性细胞的细胞制剂的免疫荧光染色和少量(<0.01%)IL-2产生T在PlaneView成像设备中的细胞。蓝色:DAPI,红色:IL-2Rα,绿色:pSTAT5,品红色:DDAO-SE。

    2. 为了测量IFNγ浓度场(图4),将10 5 SIINFEKL脉冲B16-F10黑素瘤细胞与0.2%OT-1 T细胞(200个细胞)混合并以单层沉积。然后,在顶部添加10层更多层的B16-F10细胞(10 <6>)。一个半透膜被放置在细胞上,以在进一步加工过程中保持它们的位置。


      图4.在IFNγ生产者周围的pSTAT1分布如程序D中所述制备含有掺有少量OT-1细胞的SIINFEKL脉冲B16-10A细胞的细胞制品的免疫荧光染色。蓝色:DAPI,红色:pSTAT1,绿色:DDAO-SE。

    3. 为了控制背景转录因子磷酸化和细胞因子特异性,装置装载有不含生产者的消耗细胞培养物。然后用新鲜培养基或含有10nM细胞因子的培养基覆盖细胞,分别作为阴性和阳性对照。这些样品显示阴性对照没有信号,阳性对照没有明亮,均匀的信号(Oyler-Yaniv等人,2017)。
    4. 该系统在37℃孵育1小时。之后,小心吸出培养基并将细胞在37℃下在4%PFA中固定20分钟。然后除去PFA,通过使用200μl冰冷的90%MeOH 10分钟使细胞透化以允许细胞内免疫染色。在这个阶段应该给予特别的关注,以最小程度地破坏细胞团。
    5. 固定和透化后,使用镊子将PDMS孔和膜去除。在这个阶段,细胞将紧紧地结合到载玻片上,并且可以使用标准的免疫染色方案。在室温下,在PBS中的5%FBS和0.3%Triton X-100中孵育1小时,封闭非特异性抗体结合。在室温下将一抗(兔抗-pSTAT5 1:200)在湿室中施用1小时。在室温下应用荧光团缀合的抗体(山羊抗兔Alexa 488,大鼠抗IL-2RαR-PE,1:300)1小时。然后用DAPI短暂染色细胞,并使用Fluoromount(图1B)安装盖玻片进行荧光成像。

数据分析

图像通过细分个体细胞进行处理(Gonzalez and Woods,2007),然后根据IL-2Rα表达确定每个细胞是否为消费者。对每个消费者,计算并记录pSTAT5表达的总水平和质心。这个过程的一个例子如图5所示。然后生成单个细胞上pSTAT5表达的图谱,作为离最近生产者距离的函数(图6)。


图5.图像处理程序


图6. pSTAT5分布的分析。 :一种。如程序D中所述制备掺有少量产生IL-2的细胞的100%消耗IL-2的T细胞的样品。基于总pSTAT5和每个细胞的质量中心的图像重建。每个圆的半径与pSTAT5电平成正比。 C.pSTAT5特征作为离细胞因子生产者的距离的函数。

食谱

  1. 完成RPMI
    RPMI 1640媒体补充:
    热灭活10%胎牛血清
    2 mM L-谷氨酰胺
    10 mM HEPES
    0.1 mM非必需氨基酸
    1 mM丙酮酸钠
    100微克/毫升青霉素
    100μg/ ml链霉素
    50μMβ-巯基乙醇

致谢

这项工作是与GrégoireAltan-Bonnet(NIH)合作完成的,我们要感谢他们的全面支持,特别是对于稿件的批判性阅读。我们也感谢美国和以色列的两国科学基金会(批准号为2012327,授予G.Altan-Bonnet和O.K.)的资金。该协议已被改编自Oyler-Yaniv et。,2017年。
作者声明不存在利益冲突或利益冲突。

参考

  1. Alves,NL,Richard-Le Goff,O.,Huntington,ND,Sousa,AP,Ribeiro,VS,Bordack,A.,Vives,FL,Peduto,L.,Chidgey,A.,Cumano,A.,Boyd, R.,Eberl,G。和Di Santo,JP(2009)。 表征胸腺IL-7生态位在体内。 Proc Natl Acad Sci USA 106(5):1512-1517。
  2. Böyum,A.(1968)。 隔离来自人血液的单核细胞和粒细胞。通过一次离心分离单核细胞,并通过在1g离心和沉降组合离心分离粒细胞。 Scand J Clin Lab Invest Suppl 97:77-89。
  3. Busse,D.,de la Rosa,M.,Hobiger,K.,Thurley,K.,Flossdorf,M.,Scheffold,A。和Hofer,T。(2010)。 竞争性反馈环形成了细胞微环境中辅助细胞和调节性T淋巴细胞之间的IL-2信号传导。 美国国家科学院院刊 107(7):3058-3063。
  4. Feinerman,O.,Jentsch,G.,Tkach,K. E.,Coward,J. W.,Hathorn,M. M.,Sneddon,M. W.,Emonet,T.,Smith,K. A.和Altan-Bonnet,G.(2010)。 效应细胞和调节性T细胞对IL-2反应的单细胞定量揭示了免疫应答的关键可塑性。 Mol Syst Biol 6:437。
  5. Gonzalez,R.C和Woods,R.E。(2007)。数字图像处理(第3版)。
  6. Höfer,T. O. Krichevsky,O和Altan-Bonnet,G。(2012)。 针对调节性和效应性T细胞对凿子免疫反应的IL-2竞争。 Front Immunol 3:268.
  7. Liu,Z.,Gerner,M.Y.,Van Panhuys,N.,Levine,A.G.,Rudensky,A.Y。和Germain,R.N。(2015)。 通过共定位的效应子和调节性T细胞强化免疫稳态。 Nature 528(7581):225-230。
  8. Müller,A.J.,Filipe-Santos,O.,Eberl,G.,Aebischer,T.,Spath,G.F和Bousso,P。(2012)。 CD4 + T细胞依赖细胞因子梯度来控制超出抗原呈递位点的细胞内病原体。“ CD4 + T细胞依赖细胞因子梯度来控制超出抗原呈递位点的细胞内病原体。“目标=“_ blank”> + T细胞依赖细胞因子梯度以控制超出抗原呈递位点的细胞内病原体。 免疫 37(1):147-157。
  9. Oyler-Yaniv,A.,Oyler-Yaniv,J.,Whitlock,B.M.,Liu,Z.,Germain,R.N.,Huse,M.,Altan-Bonnet,G。和Krichevsky,O。(2017)。 细胞因子增殖的可调节扩散消耗机制使免疫中细胞间通讯的可塑性成为可能系统。 免疫 46(4):609-620。
  10. Thurley,K.,Gerecht,D.,Friedmann,E。和Hofer,T。(2015)。 三在T细胞之间的细胞因子信号传导的三维梯度。
  11. Tokoyoda,K.,Egawa,T.,Sugiyama,T.,Choi,B.I。和Nagasawa,T。(2004)。 在发育期间控制骨髓内B淋巴细胞行为的细胞龛。 免疫 20(6):707-718。
  12. Weist,B.M.,Kurd,N.,Boussier,J.,Chan,S.W。和Robey,E.A。(2015)。 胸腺调节性T细胞生态位大小是通过限制带有抗原的树突状细胞的IL-2和反馈来规定的竞争。 Nat Immunol 16(6):635-641。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Oyler-Yaniv, A. and Krichevsky, O. (2018). Imaging Cytokine Concentration Fields Using PlaneView Imaging Devices. Bio-protocol 8(7): e2788. DOI: 10.21769/BioProtoc.2788.
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