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Apr 2019

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Optogenetic Tuning of Ligand Binding to The Human T cell Receptor Using The opto-ligand-TCR System
利用光配体TCR系统调节配体与人T细胞受体的结合   

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Abstract

T cells are one major cell type of the immune system that use their T cell antigen receptor (TCR) to bind and respond to foreign molecules derived from pathogens. The ligand-TCR interaction half-lives determine stimulation outcome. Until recently, scientists relied on mutating either the TCR or its ligands to investigate how varying TCR-ligand interaction durations impacted on T cell activation. Our newly created opto-ligand-TCR system allowed us to precisely and reversibly control ligand binding to the TCR by light illumination. This system uses phytochrome B (PhyB) tetramers as a light-regulated TCR ligand. PhyB can be photoconverted between a binding (ON) and non-binding (OFF) conformation by 660 nm and 740 nm light illumination, respectively. PhyB ON is able to bind to a synthetic TCR, generated by fusing the PhyB interacting factor (PIF) to the TCRβ chain. Switching PhyB to the OFF conformation disrupts this interaction. Sufficiently long binding of PhyB tetramers to the PIF-TCR led to T cell activation as measured by calcium influx. Here, we describe protocols for how to generate the tetrameric ligand for our opto-ligand-TCR system, how to measure ligand-TCR binding by flow cytometry and how to quantify T cell activation via calcium influx.

Keywords: Optogenetic (光遗传学), T cell receptor (T细胞受体), Reversible ligand binding (可逆配体结合), Opto-ligand-TCR (Opto-ligand-TCR), Spatiotemporal control (实时原位控制)

Background

Life depends to a large extent on the precise spatial and temporal coordination of molecular events. This is particularly important in cellular decision processes, for which cells constantly interpret signals from their environment in order to decide how to respond. Due to the lack of appropriate approaches, the impact of kinetics and localization of signaling processes on cellular decisions is still not well understood. Now the emerging field of optogenetics enables to perform the experiments required to fill this knowledge gap (Kolar and Weber, 2017; Goglia and Toettcher, 2019). As an example, we use T cells stimulated via their T cell antigen receptor (TCR) in this protocol.

T cells are a crucial part of the adaptive immune system. Beyond their role in protecting the body from infections, T cells have recently gotten attention for their potential in cancer immunotherapy. Hence, a better understanding of the mechanisms behind T cell activation is highly wanted (Spear et al., 2019). Using their TCR, T cells can sense foreign particles, such as viruses and bacteria. Those particles are recognized by the TCR in the form of pathogen-derived peptides presented on major histocompatibility complex (MHC) proteins. These peptide-MHC conjugates serve as high affinity ligands for the TCR (Davis et al., 1998). Importantly, self peptides derived from endogenous proteins are presented on MHC as well. Self peptide-MHCs also bind to the TCR, but with low affinity and thus do not result in activatory signaling. It is therefore clear that TCRs are able to distinguish between ligands of different affinity (Holler and Kranz, 2003) and it has been proposed that T cells are able to make this differentiation based on the ligand binding time to the TCR (McKeithan, 1995).

So far, the majority of T cell researchers used peptides with point mutations, presented on MHC to investigate the effect of varying ligand affinity (and indirectly binding time) on T cell activation (Matsui et al., 1991 and 1994; Weber et al., 1992; Sykulev et al., 1994; Corr et al., 1994; Lyons et al., 1996; Daniels et al., 2006). Alternative methods for changing ligand-TCR interaction time are the use of mutated superantigens (Andersen et al., 2001) or the mutation of the TCR itself (Tan et al., 2017) All those approaches have in common that they fail to exclusively manipulate the TCR-ligand binding time without affecting other properties of the binding event, such as on-rate, enthalpy, entropy, geometry of binding, Gibbs energy or ability to withstand forces.

To overcome these experimental restrictions we have developed the opto-ligand-TCR system (Yousefi et al., 2019), by making use of the light-dependent interaction between phytochrome B (PhyB) and PhyB interacting factor (PIF) (Levskaya et al., 2009; Toettcher et al., 2013; Kolar and Weber, 2017). We chose the PhyB-PIF system as the optogenetic switch for our system, since it allows for active, light-dependent conformational changes in both directions on a timescale of milliseconds to seconds. On the basis of our expertise to engineer and work with TCRs (Minguet et al., 2007; Swamy et al., 2016; Baeuerle et al., 2019; Schamel et al., 2019), we fused a PIF optimized for the secretory pathway (PIFS) together with a monomeric green fluorescent protein (GFP) to the TCRβ chain. This synthetic GFP-PIFS-TCRβ was expressed as part of the complete TCR complex on the surface of Jurkat T cells (Figure 1). Biotinylated PhyB molecules were tetramerized via streptavidin and these PhyB tetramers (PhyBt) were used as multimeric TCR ligands. 660 nm light illumination of PhyB led to a switch to the PIF-binding ON state (usually referred to as Pfr state) and 740 nm illumination reverses PhyB to the non-binding OFF state (usually referred to as Pr state) (Mancinelli, 1994). Hence, our opto-ligand-TCR system enabled us to specifically control ligand binding times via light illumination using the same ligand-receptor pair and without introducing mutations to the TCR or its ligands.

Our novel system allows high spatiotemporal control over reversible ligand binding to the TCR. This unique feature of the opto-ligand-TCR system could enable researchers to locally or timely restrict ligand-receptor interaction. Fusing PIF to other receptors would allow to control ligand binding to those receptors as well, as we previously demonstrated for integrins (Baaske et al., 2019). Further, our system could be used to investigate the signaling events that happen after ligand dissociation, which have been mostly neglected due to the lack of appropriate methods.


Figure 1. Scheme of the opto-ligand-TCR system. Phytochrome B (PhyB) tetramers can be switched between a binding ON and a non-binding OFF state via 660 nm or 740 nm light illumination, respectively. PhyBt ON are able to bind to Jurkat T cells expressing GFP-PIFS-TCR on the surface, thereby activating downstream signaling, part of which is the influx of calcium ions.

Materials and Reagents

  1. Pipette tips 0.1-20 µl (VWR, catalog number: 613-1067 )
  2. Pipette tips 1-200 µl (Carl Roth, catalog number: 7058 )
  3. Pipette tips 100-1,200 µl (Ratiolab, catalog number: 2400610 )
  4. 0.22 µm syringe filters (GE Healthcare, Whatman, catalog number: 10462200 )
  5. 1 ml and 5 ml syringes (Terumo, catalog numbers: SS+01T1 and SS*05LE1 )
  6. 1.5 ml reaction tubes (Sarstedt, catalog number: 72.690.001 )
  7. 15 ml and 50 ml conical tubes (Greiner Bio-One, catalog numbers: 188271 and 227261 )
  8. 3.5 ml FACS tubes (Sarstedt, catalog number: 55.484 )
  9. HiLoad Superdex 200 pg column (GE Healthcare, catalog number: 28989335 ), store at 4 °C
  10. Jurkat GFP-PIFS-TCR cells (Yousefi et al., 2019)
  11. Streptavidin, DyLight650-conjugated (Thermo Fisher, Invitrogen, catalog number: 84547 ), store at 4 °C; molecular weight is approximately 53 kDa
  12. Dulbecco’s phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number D8537 ), store at 4 °C
  13. Tris-(2-carboxyethyl)-phosphine (TCEP) hydrochloride (Carl Roth, catalog number: HN95 ), store at 4 °C
  14. NaN3 (Carl Roth, catalog number: K305 ), store at room temperature
  15. Indo-1 (Thermo Fisher, Invitrogen, catalog number: I1223 ), store at 4 °C
  16. Pluronic F-127 (Thermo Fisher, Invitrogen, catalog number: P3000MP ), store at room temperature
  17. Roswell Park Memorial Institute (RPMI) medium (US Biologicals, catalog number: R9002 ), store powder at room temperature, store dissolved medium at 4 °C
  18. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number F7524 ), store at 4 °C
  19. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Thermo Fisher, catalog number: 15630-080 ), store at 4 °C
  20. Purified PhyB-AviTag monomers (Hörner et al., 2020, store at -80 °C; molecular weight is approximately 74 kDa
  21. NaOH pellets (Merck, catalog number: 1064981000 )
  22. 10% NaN3 (see Recipes)
  23. 5 M NaOH (see Recipes)
  24. 0.5 M TCEP (see Recipes)
  25. Protein buffer (see Recipes)
  26. FACS buffer (see Recipes)
  27. Stimulation medium (see Recipes)

Equipment

  1. Pipettes (Eppendorf, catalog number: 3123000900 )
  2. ÄKTAexplorer 10S (GE Healthcare, ÄKTAexplorer 10S)
  3. MACSQuant X Flow Cytometer, customized with 20 mW 355 nm laser and 405/20 nm as well as 530/30 nm emission bandpass filters (Miltenyi Biotec)
  4. Centrifuge 5810 R (Eppendorf)
  5. Incubator HeraCell 150i (Thermo Fisher)
  6. Sterile hood Safe 2020 (Thermo Fisher)
  7. pxONE equipped with 660 nm and 740 nm LEDs (Opto Biolabs)
  8. Green safe light: Deco flex RGB LED strip, set to green light (Osram, catalog number: 76123 )

Software

  1. Unicorn 5.11 (GE Healthcare)
  2. FlowJo 9 (Tree Star Inc.)
  3. Prism 6 (GraphPad Software Inc.)
  4. Illustrator CC (Adobe Inc.)

Procedure

  1. Phytochrome B tetramer (PhyBt) production
    1. Purify biotinylated phytochrome B (PhyB-AviTag) monomers as described in Hörner et al., 2020.
    2. Mix a 10-fold molar excess of PhyB-AviTag monomers at a concentration between 1 and 5 mg/ml with DyLight650-conjugated streptavidin in protein buffer for a total volume of 2.5 ml and incubate either for 2 h at room temperature or overnight at 4 °C and in the dark.
    3. Separate PhyB tetramers (PhyBt) from the excess of PhyB-AviTag monomers via size exclusion chromatography using a HiLoad Superdex 200 pg column on an ÄKTAexplorer 10S chromatography system.
      1. Perform all steps in a cold room at 4 °C and minimize protein exposure to light. Use a flow rate of 1 ml/min.
      2. Equilibrate the column with two column volumes freshly prepared protein buffer.
      3. Load the protein mixture on the column using a 2 ml sample loop and run for two column volumes while collecting 1 ml fractions.
      4. Protein elution can be monitored by the absorbance at 280 nm (A280), PhyB and DyLight650-conjugated streptavidin by the absorbance at 650 nm (A650) and PhyB monomers and tetramers by the absorbance at 365 nm (A365). See Figure 2 for an example of a purification run.
      5. Pool all PhyBt-containing fractions and determine the PhyBt concentration by the ΔΔA method:
        1. Perform a spectral analysis of the purified PhyBt as described for PhyB-AviTag monomers in Hörner et al., 2020 under procedure C3.
        2. Calculate the ΔΔA value by subtracting the minimum value at 711 nm from the maximum value at 649 nm of the difference spectrum.
        3. Multiply the ΔΔA value with 1.1 to get the PhyB concentration in mg/ml. The factor 1.1 was determined from ΔΔA measurements of PhyB-AviTag monomers correlated to protein concentration measurements via Bradford assay.
        4. Divide the PhyB concentration in mg/ml by the molecular weight of PhyB-AviTag of 74,000 g/mol to get the PhyB-AviTag concentration in M (mol/L).
        5. Divide the the PhyB-AviTag monomer concentration by 4 to get the PhyBt concentration in M.
      6. Filter the PhyBt solution using a 0.22 µm syringe filter and aliquot. Sterile aliquots can be stored up to 4 weeks at 4 °C and in the dark.
      7. This protocol usually yields 80-90% of the used DyLight650-conjugated streptavidin as PhyBt at a concentration between 0.5 and 3 µM.


        Figure 2. Purification of PhyB tetramers (PhyBt) from PhyB monomers via size exclusion chromatography. PhyB bound to DyLight650-conjugated streptavidin was separated from the excess of PhyB monomers on a HiLoad Superdex 200 pg column. Total protein absorbance was monitored at 280 nm, the absorbance of PhyB was detected at 365 nm and the combined absorbance of PhyB and DyLight650 was followed at 650 nm. Results show one experiment of n > 3.

  2. Light-dependent PhyBt binding to the GFP-PIFS-TCR on the T cell surface
    1. Cultivate Jurkat GFP-PIFS-TCR cells according to standard Jurkat cell culture conditions (see e.g., Yousefi et al., 2019). The cells should be kept at a density between 0.5 and 1.0 million cells per ml prior to the experiment.
    2. Transfer 3 x 105 Jurkat GFP-PIFS-TCR cells for each sample into FACS tubes.
    3. Centrifuge the samples for 4 min at 300 x g at 4 °C.
    4. Perform the following steps on ice (or even better in a 4 °C room) with cold buffers.
    5. Discard the supernatant by aspiration or decanting and resuspend cells in 1 ml FACS buffer.
    6. Repeat the centrifugation and supernatant removal steps as above.
    7. During the centrifugation steps, dilute PhyBt to a final concentration of 100 nM in FACS buffer. The total volume of diluted PhyBt solution depends on the number of samples, with 50 µl being needed per sample.
    8. Divide the PhyBt solution into two and illuminate one half with saturating amounts of 660 nm light, resulting in PhyBt(660), and the other half with saturating amounts of 740 nm light, resulting in PhyBt(740). An illumination for 5 min at 100 µmol/m2s is sufficient.
    9. The following handling steps should be performed under green safe light to prevent PhyB photoconversion. It is essential to prevent any white light (sunlight or room light) from hitting the samples.
    10. Resuspend the cell samples in 50 µl of either PhyBt(660) or PhyBt(740) and incubate for 30 min on ice and in the dark.
    11. Wash the samples two times as described under Steps B3-B5.
    12. Resuspend the cells in 200 µl FACS buffer and measure the PhyBt (DyLight650) fluorescence in a flow cytometer. Representative results are depicted in Figure 3.


      Figure 3. Measurement of PhyBt binding to the GFP-PIFS-TCR on the T cell surface. A. Jurkat GFP-PIFS-TCR cells were treated with PBS (grey), PhyBt(740) (red) or PhyBt(660) (orange) and surface binding of PhyBt was measured by flow cytometry via DyLight650 fluorescence. Results show one experiment of n > 3 (a.u., arbitrary units). B. Median fluorescence intensity (MFI, blue) and percent of PhyBt-bound cells (green) were quantified as measured in (A). Only PhyBt(660) showed considerable binding to the T cells. Results depict the mean of duplicate measurements ± SD of one experiment out of n > 3.

  3. Analysis of T cell activation via Ca2+ influx upon light-dependent PhyBt binding
    1. Transfer 5 x 106 freshly dividing Jurkat GFP-PIFS-TCR cells into a 15 ml conical tube.
    2. Centrifuge the cells for 4 min at 300 x g at room temperature.
    3. Discard the supernatant by aspiration and resuspend the cells in 1 ml stimulation medium.
    4. The cells should be resuspended by gentle pipetting and never by vortexing as this can already lead to cell activation and impair the Ca2+ influx measurements.
    5. Repeat the centrifugation and supernatant removal as above.
    6. Prepare the Indo-1 staining solution during the centrifugation step by mixing 5 µl Pluronic F-127 with 4 µl Indo-1 and 1 ml stimulation medium.
    7. Resuspend the cells in 1 ml staining solution, transfer the cell suspension into a 1.5 ml reaction tube and incubate for 15 min in a cell culture incubator keeping the lid open.
    8. After 15 min incubation, close the lid of the reaction tube, briefly invert the tube 5-10 times and incubate the cells for another 15 min.
    9. Repeat centrifugation and supernatant removal as in Steps C2 and C3 and resuspend the cells in 500 µl 4 °C cold stimulation medium.
    10. Keep the cells on ice and in the dark for 15 min before starting the measurements.
    11. For each measurement, freshly prepare 1 ml diluted cell suspension by gently mixing 50 µl of the stained cells with 950 µl 37 °C pre-warmed stimulation medium in a FACS tube.
    12. Transfer the FACS tube into the pxONE incubation and illumination device and insert the device into the flow cytometer. Details about the use of the pxONE device can be found on the manufacturer’s website (www.optobiolabs.com) and a video article providing a step-by-step explanation of a prototype is available (Brenker et al., 2016).
    13. Measure 1 min baseline fluorescence, then add between 2 and 200 nM of either PhyBt(660) or PhyBt(740) and measure another 5 min in the dark. Figure 4 depicts an example of such an experiment.
    14. Vary the PhyBt concentration and the illumination conditions to your specific experimental question. Examples of different lighting regimes for various experimental questions can be found in Yousefi et al., 2019.


      Figure 4. Calcium influx measurement upon light-dependent stimulation with the opto-ligand-TCR system. A. Calcium influx into Jurkat GFP-PIFS-TCR cells was measured via Indo-1 fluorescence upon addition of 20 nM PhyBt(740) or PhyBt(660) as indicated. The stimulation was performed in the dark and stimuli addition is indicated by the arrow. Results show one experiment of n > 3. B. The percent of responding cells were quantified from (A) as described in the Data Analysis section. Results show one experiment of n > 3. T cells treated with PhyBt(660) showed calcium influx that peaked at around 250 s as opposed to PhyBt(740)-treated cells.

Data analysis

The Unicorn software suite was used to control the Äkta chromatography system and analyze the resulting data. FlowJo was used to analyze all flow cytometry data. Only the living cell population was used for the depicted flow cytometry results. Intracellular calcium was quantified by the ratio of Ca2+-bound Indo-1 (405/20 nm filter) and Ca2+-free Indo-1 (530/30 nm filter). The percentage of responding cells shown in Figure 4A were derived from FlowJo’s kinetics module using the 90th percentile over the baseline measurement between 30 and 60 s. For Figures 2, 3B and 4B, quantified data were exported from Unicorn and FlowJo, respectively, and displayed using Prism. Figures 3A and 4A were directly exported from FlowJo. All figures were compiled using Illustrator.

Notes

  1. Protect PhyB from bright light, like white room light or direct sunlight, by covering it with aluminum foil or dimming the room, if possible. Whenever the conformation of PhyB should not be changed, work under green safe light, which lies at the absorbance minimum of PhyB. White room light or sunlight has a similar effect as 660 nm illumination and converts PhyB to the ON state.
  2. PhyB is sensitive to oxidation and therefore should be kept in a reducing buffer by for example degassing solutions and adding 0.5 mM TCEP, β-mercaptoethanol or dithiothreitol. Whenever the experimental conditions prohibit the use of reducing agents, expect and monitor a decline in PhyB to PIF binding activity over the course of several hours.
  3. Continuous 660 nm light illumination may prevent PhyBt from binding to the GFP-PIFS-TCR as described in Yousefi et al. (2019). Therefore, use either low 660 nm light intensities or pulsed illumination with dark periods in between. 660 nm light illumination of increasing intensity results in shorter PhyBt binding half-lives to the GFP-PIFS-TCR.
  4. Due to the photobiology of PhyB, saturating illumination with our 660 nm light source resulted in 80% of the molecules in the PhyB ON state and 20% in the PhyB OFF state. Saturating illumination with our 740 nm light source gave 1% PhyB ON and 99% PhyB OFF. Different light sources with altering emission spectra will result in different PhyB ON:OFF ratios, for details see Smith et al. (2016).

Recipes

  1. 10% NaN3
    1. Dissolve NaN3 in water
    2. Store at room temperature
  2. 5 M NaOH
    1. Dissolve NaOH pellets in water by adding the pellets slowly to the water (exothermic reaction)
    2. Store at room temperature
  3. 0.5 M TCEP
    1. Dissolve TCEP in degassed water
    2. Adjust pH to 7.4 with 5 M NaOH
    3. Store in 1 ml aliquots at -20 °C
  4. Protein buffer
    1. PBS
    2. Add 0.5 mM TCEP from 0.5 M stock solution
    3. Run through 0.22 µm filter and degas
    4. Do not store protein buffer, but always prepare freshly
  5. FACS buffer
    1. PBS
    2. 1% FBS
    3. 0.02% NaN3 (dilute from 10% stock)
    4. Store at 4 °C
  6. Stimulation medium
    1. RPMI
    2. 1% FBS
    3. 10 mM HEPES
    4. Store at 4 °C

Acknowledgments

We thank Susan Lauw and Johannes Kaiser from the Signalhaus Robotics Facility for running of the robotics platform (INST 39/899–1 FUGG) to conduct the calcium experiment as well as Opto Biolabs for their customized illumination devices. This work was funded by the Deutsche Forschungsgemeinschaft (DFG) under Germany’s Excellence Strategy through EXC294 (BIOSS–Center for Biological Signalling Studies), EXC2189 (CIBSS–Centre for Integrative Biological Signalling Studies, Project ID 390939984), SFB854 (B19), SCHA976/7-1, SCHA976/8-1 and Project-ID 403222702 - SFB 1381 (A9). The procedure described in detail in this protocol paper was derived from our article in eLife (Yousefi et al., 2019).

Competing interests

Opto Biolabs provided us with the pxONE device.

References

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  27. Yousefi, O. S., Gunther, M., Hörner, M., Chalupsky, J., Wess, M., Brandl, S. M., Smith, R. W., Fleck, C., Kunkel, T., Zurbriggen, M. D., Hofer, T., Weber, W. and Schamel, W. W. (2019). Optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor. Elife 8: e42475.

简介

[摘要] T细胞是免疫系统的一种主要细胞类型,利用其T细胞抗原受体(TCR)结合并响应源自病原体的外来分子。配体-TCR相互作用的半衰期决定了刺激的结果。直到最近,科学家还是依靠突变TCR或其配体来研究变化的TCR-配体相互作用持续时间如何影响T细胞活化。我们新创建的光配体-TCR系统使我们能够通过光照精确且可逆地控制配体与TCR的结合。该系统使用植物色素B(PhyB )四聚体作为光调节的TCR配体。PHYB 可光转化的结合(ON)之间,并且通过分别为660nm和740nm的光照射,非结合性(OFF)构象。PhyB ON能够结合通过将PhyB 相互作用因子(PIF)融合到TCRβ链而产生的合成TCR 。将PhyB 切换为OFF构象会破坏这种相互作用。PhyB 四聚体与PIF-TCR 足够长的结合导致T细胞活化(通过钙内流测量)。在这里,我们描述了有关如何为我们的光配体-TCR系统生成四聚体配体,如何通过流式细胞仪测量配体-TCR结合以及如何通过钙内流量化T细胞活化的协议。

[背景 ] 寿命很大程度上依赖于分子事件的精确时间和空间协调很大程度上。这在细胞决策过程中尤为重要,因为细胞不断地解释其周围环境的信号以决定如何响应。由于缺乏适当的方法,动力学和信号转导过程的本地化对细胞决定的影响仍然不是很了解。现在光遗传学的新兴领域使人们能够进行填补这一知识空白所需的实验(Kolar和Weber,2017年; Goglia和Toettcher,201 9 )。例如,在此方案中,我们使用通过T细胞抗原受体(TCR)刺激的T细胞。

T细胞是适应性免疫系统的关键部分。T细胞除了具有保护人体免受感染的作用外,最近还因其在癌症免疫疗法中的潜力而受到关注。因此,高度希望更好地理解T细胞活化背后的机制(Spear 等,2019)。T细胞使用TCR可以感知外来颗粒,例如病毒和细菌。TCR会以主要组织相容性复合体(MHC)蛋白上呈现的病原体衍生肽的形式识别这些颗粒。这些肽-MHC缀合物充当TCR的高亲和力配体(Davis 等,1998)。重要的是,源自内源蛋白的自身肽也存在于MHC上。自身肽-MHC也与TCR结合,但亲和力低,因此不会导致激活信号传导。因此,很明显,TCR能够区分具有不同亲和力的配体(Holler和Kranz,2003),并且已经提出T细胞能够基于配体与TCR的结合时间进行这种分化(McKeithan,1995)。。

到目前为止,大多数T细胞研究人员使用了在MHC上呈递的具有点突变的肽,以研究变化的配体亲和力(和间接结合时间)对T细胞活化的影响(Matsui 等,1991 和1994; Weber 等。,1992; Sykulev 等,1994; Corr 等,1994; Lyons 等,1996; Daniels 等,2006)。改变配体-TCR相互作用时间的替代方法是使用突变的超抗原(Andersen 等,2001)或TCR本身的突变(Tan 等,2017)。所有这些方法的共同点是它们无法完全操纵TCR-配体的结合时间,而不会影响结合事件的其他性质,例如接通速率,焓,熵,结合几何形状,吉布斯能量或承受力的能力。

为了克服这些实验限制,我们通过利用植物色素B(PhyB )和PhyB 相互作用因子(PIF)之间的光依赖相互作用,开发了光配体TCR系统(Yousefi 等,2019 )(Levskaya 等。,2009; Toettcher 。等人,2013;科拉尔和Weber,2017) 。我们选择PhyB -PIF系统作为系统的光遗传学开关,因为它允许在毫秒到秒的时间范围内在两个方向上进行主动的,依赖于光的构象更改。根据我们在设计和使用TCR 方面的专业知识(Minguet 等人,2007; Swamy 等人,2016 ; Baeuerle 等人,2019; Schamel 等人,2019),我们融合了针对TCR 优化的PIF分泌途径(PIF S )和单体绿色荧光蛋白(GFP)一起到达TCRβ链。这种合成GFP-PIF 小号-TCR β 表示为的表面上的完整TCR复合物的一部分的Jurkat T细胞(图1)。通过链霉亲和素将生物素化的PhyB 分子四聚化,并将这些PhyB 四聚体(PhyBt )用作多聚TCR配体。PhyB的660 nm光照导致切换到PIF绑定打开状态(通常称为Pfr 状态),而740 nm光照使PhyB 反转为非绑定关闭状态(通常称为Pr 状态)(Mancinelli,1994年) )。因此,我们的光配体-TCR系统使我们能够使用相同的配体-受体对,通过光照特异性控制配体结合时间,而不会在TCR或其配体上引入突变。

我们的新型系统允许对可逆配体与TCR的结合进行高度的时空控制。光配体-TCR系统的这一独特功能可使研究人员局部或及时限制配体-受体的相互作用。将PIF与其他​​受体融合也可以控制配体与那些受体的结合,正如我们之前对整联蛋白所证明的那样(Baaske et al。,2019)。此外,我们的系统可用于研究配体解离后发生的信号传递事件,由于缺乏适当的方法,这些事件已被忽略。



D:\ Reformatting \ 2020-1-6 \ 1902676--1297 Wolfgang Schamel 784708 \ Figs jpg \ Fig1.jpg

图1 。光学配体-TCR系统的方案。植物色素B(PhyB )四聚体可以分别通过660 nm或740 nm的光照在结合ON状态和非结合OFF状态之间切换。PhyBt ON能够与表面表达GF P-PIF S -TCR的Jurkat T细胞结合,从而激活下游信号传导,其中一部分是钙离子的流入。

关键字:光遗传学, T细胞受体, 可逆配体结合, Opto-ligand-TCR, 实时原位控制

材料和试剂


 


P ipette提示0.1-20微升(VWR,目录号:613-1067)
移液器吸头1-200 µl(Carl Roth,目录号:7058)
移液管头100-1 ,200微升(Ratiolab ,目录号:2400610)
0.22 µm注射器过滤器(GE Healthcare,Whatman,目录号:10462200)
1 ml和5 ml注射器(Terumo,目录号:SS + 01T1和SS * 05LE1)
1.5 ml反应管(Sarstedt ,目录号:72.690.001)
15毫升和50ml锥形管(格雷纳乙IO- ø NE,产品目录号:188271和227261)
3.5 ml FACS管(Sarstedt ,目录号:55.484)
HiLoad Superdex 200 pg 色谱柱(GE Healthcare,目录号:28989335),在4°C下储存
Jurkat GFP-PIF S -TCR细胞(Yousefi et al。,2019)
链霉亲和素,与DyLight650偶联(Thermo Fisher,Invitrogen,目录号:84547),储存在4°C下;米olecular重量约为53 kDa的
Dulbecco的磷酸盐缓冲盐水(PBS) (Sigma-Aldrich,货号D8537),储存在4°C
三(2-羧乙基)膦(TCEP)盐酸盐(Carl Roth,目录号:HN95),在4°C下储存
NaN 3 (Carl Roth,目录号:K305),在室温下保存
Indo-1(Thermo Fisher,Invitrogen,目录号:I1223),存储在4°C下
Pluronic F-127(Thermo Fisher,Invitrogen,目录号:P3000MP),在室温下存储
罗斯威尔公园纪念研究所(RPMI)介质(美国生物制药,目录号:R9002),在室温下存储粉末,在4°C下存储溶解介质
胎牛血清(FBS)(Sigma-Aldrich,目录号F7524),在4°C下储存
4-(2-羟乙基)-1-哌嗪乙烷磺酸(HEPES)(Thermo Fisher,目录号:15630-080),在4°C下储存
纯化PHYB-shRNA表达克隆单体(霍纳等人,2020 ,储存在-80℃下; 米olecular 重量约为74 kDa的
NaOH颗粒(Merck,目录号:1064981000)
10%NaN 3 (请参阅食谱)
5 M NaOH(请参阅食谱)
0.5 M TCEP(请参阅食谱)
蛋白质缓冲液(请参阅食谱)
FACS缓冲区(请参阅配方)
刺激介质(请参见食谱)
 


设备


 


小管(埃彭多夫(Eppendorf),目录号:3123000900)
ÄKTAexplorer 10S (GE医疗集团,ÄKTAexplorer 10S )
MACSQuant X流式细胞仪,使用20 mW 355 nm激光和405/20 nm以及530/30 nm发射带通滤光片(Miltenyi Biotec )定制
5810 R离心机(Eppendorf)
孵化器HeraCell 150i (Thermo Fisher)
无菌罩Safe 2020(Thermo Fisher)
配备660 nm和740 nm LED的pxONE (Opto Biolabs )
绿色安全灯:Deco flex RGB LED灯带,设置为绿色灯(欧司朗,目录号:76123)
 


软件


 


独角兽5.11(GE Healthcare)
的FlowJo 9(树星公司。)
棱镜6(格拉夫派得软件公司。)
插画CC(的Adobe公司。)




程序


 


植物色素B四聚体(PhyBt )的生产
按照Hörner 等人的方法纯化生物素化的植物色素B(PhyB-AviTag )单体。,2020年。
在蛋白质缓冲液中混合10倍摩尔过量的PhyB-AviTag 单体(浓度为1-5 mg / ml)与DyLight650偶联的抗生蛋白链菌素,将其总体积为2.5 ml,并在室温下孵育2 h或在4 下孵育过夜°C 和黑暗中。
使用ÄKTAexplorer10S 色谱系统上的HiLoad Superdex 200 pg 色谱柱,通过尺寸排阻色谱,从过量的PhyB-AviTag 单体中分离出PhyB 四聚体(PhyBt )。
P erform在4℃下在冷室中的所有步骤,并尽量减少蛋白暴露于光。使用1 ml / min的流速。
用两倍柱体积的新鲜制备的蛋白质缓冲液平衡柱。
使用2 ml样品定量环将蛋白质混合物上样到色谱柱上,并运行两个色谱柱体积,同时收集1 ml馏分。
可以通过280 nm(A 280 )的吸光度监测蛋白质洗脱,通过650 nm(A 650 )的吸光度监测PhyB 和DyLight650共轭链霉亲和素,通过365 nm(A 365 )的吸光度监测PhyB 单体和四聚体。有关纯化运行的示例,请参见图2。
池中的所有PhyBt 含级分,并确定PhyBt 由浓度ΔΔ 的方法:
按照Hörner 等人对PhyB-AviTag 单体所述,对纯化的PhyBt 进行光谱分析。,程序2020中的3。
通过从差分光谱的649 nm处的最大值减去711 nm处的最小值来计算ΔΔA 值。
将ΔΔA 值乘以1.1得到以mg / ml 为单位的PhyB 浓度。所述因子1.1 是从确定ΔΔ 的测量PHYB-shRNA表达克隆通过Bradford测定相关蛋白浓度测量的单体。
划分PHYB 通过的分子量以mg / ml的浓度PHYB-shRNA表达克隆74,000克/ 摩尔,以获得PHYB-shRNA表达克隆以M浓度(mol / 大号)。
划分的PHYB-shRNA表达克隆通过4单体浓度,以获得PhyBt 在M.浓度
使用0.22 µm注射器过滤器过滤等分的PhyBt 溶液。无菌等分试样可在4°C 和黑暗中最多保存4周。
该方案通常以0.5到3 µM的浓度产生80-90%的DyDY650偶联的抗生蛋白链菌素PhyBt 。
 


D:\ Reformatting \ 2020-1-6 \ 1902676--1297 Wolfgang Schamel 784708 \ Figs jpg \ Fig2.jpg


图2 。通过尺寸排阻色谱法从PhyB 单体中纯化PhyB 四聚体(PhyBt )。PHYB 势必DyLight650共轭的链亲和素是从过量的分离PHYB 单体上的的HiLoad 的Superdex 200 皮克柱。在280nm处监测总蛋白吸光度,在365nm处检测PhyB 的吸光度,并在650nm处跟踪PhyB 和DyLight650 的组合吸光度。结果显示n > 3的一项实验。


 


光依赖性PhyBt 与T细胞表面的GF P-PIF S - TCR 结合
根据标准Jurkat 细胞培养条件培养Jurkat GFP-PIF S -TCR细胞(参见例如Yousefi 等人,2019 )。实验前,细胞的密度应保持在每毫升0.5至100万个细胞之间。
将每个样品的3 x 10 5 Jurkat GFP-PIF S -TCR细胞转移到FACS管中。
在4°C下以300 xg 离心样品4分钟。
在冰上(或在4°C的房间里甚至更好)用冷缓冲液执行以下步骤。
通过抽吸或倾析丢弃上清液,并将细胞重悬于1 ml FACS缓冲液中。
重复上述离心和上清液去除步骤。
在离心步骤中,在FACS缓冲液中将PhyBt 稀释至终浓度100 nM 。稀释的PhyBt 溶液的总体积取决于样品数量,每个样品需要50 µl。
将PhyBt 溶液分成两部分,用饱和量的6 60 nm光照射一半,得到PhyBt (660),而用饱和量的7 40 nm光照射另一半,得到PhyBt (740)。以100 µmol / m 2 s 的光照5分钟就足够了。
应在绿色安全光下执行以下处理步骤,以防止PhyB 光转换。防止任何白光(阳光或室内光)照射样品至关重要。
将细胞样品重悬于50 µl PhyBt (660)或PhyBt (740)中,并在冰上和黑暗中孵育30分钟。
按照步骤B3-B5 所述将样品洗涤两次。
将细胞重悬于200 µl FACS缓冲液中,并在流式细胞仪中测量PhyBt (DyLight650 )荧光。代表性的结果见图3。
 


D:\ Reformatting \ 2020-1-6 \ 1902676--1297 Wolfgang Schamel 784708 \ Figs jpg \ Fig3.jpg


图3 。PhyBt与T细胞表面GFP-PIF S -TCR 结合的测量。一。用PBS(灰色),PhyBt (740)(红色)或PhyBt (660)(橙色)处理Jurkat GFP-PIF S -TCR细胞,并通过流式细胞仪通过DyLight650 荧光测量PhyBt的表面结合。结果显示了一个n > 3(au 。,任意单位)的实验。乙。如在(A)中所测量的,对中值荧光强度(MFI,蓝色)和与PhyBt 结合的细胞的百分比(绿色)进行定量。只有PhyBt (660)表现出与T细胞的显着结合。结果描述了n > 3 中一个实验重复测量的平均值±SD 。


 


通过光依赖的PhyBt 结合,通过Ca 2+内流激活T细胞的分析
将5 x 10 6 新鲜分裂的Jurkat GFP-PIF S -TCR细胞转移到15 ml锥形管中。
在室温下以300 xg 离心细胞4分钟。
吸除上清液,将细胞重悬于1 ml刺激培养基中。
细胞应通过轻轻移液重悬,切勿涡旋重悬,因为这可能已导致细胞活化并损害Ca 2+内流测量。
重复上述离心和去除上清液的操作。
通过将5 µl Pluronic F-127与4 µl Indo-1和1 ml刺激培养基混合,在离心步骤中准备Indo-1染色溶液。
将细胞重悬于1 ml染色溶液中,将细胞悬液转移至1.5 ml反应管中,并在细胞培养箱中孵育15分钟,保持盖子打开。
孵育15分钟后,关闭反应管的盖子,将试管短暂颠倒5-10次,再将细胞孵育15分钟。
重复离心和除去上清液如步骤小号C2和C3和重悬细胞于500μl4℃寒冷刺激平台。
在开始测量之前,将细胞在冰上和黑暗中放置15分钟。
对于每次测量,将50 µl染色的细胞与950 µl 37°C预热的刺激培养基在FACS管中轻轻混合,以新鲜制备1 ml稀释的细胞悬液。
将FACS管转移到pxONE 孵育和照明设备中,然后将设备插入流式细胞仪中。可以在制造商的网站(www.optobiolabs.com)上找到有关pxONE 设备使用的详细信息,并提供了一段视频,其中提供了有关原型的逐步说明(Brenker 等,2016)。
测量1种分钟基线荧光,然后2和200之间添加纳米ö ˚F任PhyBt (660)或PhyBt (740),测量在暗处另外5分钟。图4描绘了这样一个实验的例子。
根据您的具体实验问题改变PhyBt 浓度和光照条件。可以在Yousefi et al 。,2019中找到针对各种实验问题的不同照明方式的示例。
 


D:\ Reformatting \ 2020-1-6 \ 1902676--1297 Wolfgang Schamel 784708 \ Figs jpg \ Fig4.jpg


图4 。用光配体-TCR系统在光依赖性刺激下测量钙的流入量。一。如图所示,在加入20 nM PhyBt (740)或PhyBt (660)后,通过Indo-1荧光测量流入Jurkat GFP-PIF S -TCR细胞的钙流入量。刺激是在黑暗中进行的,刺激的添加如箭头所示。结果显示了一个n > 3的实验。B 。如数据分析部分所述,从(A)中定量反应细胞的百分比。结果显示,n > 3的一项实验。与PhyBt (740)处理的细胞相比,用PhyBt (660)处理的T细胞显示钙流入在250 s左右达到峰值。


 


数据分析


 


Unicorn软件套件用于控制Äkta 色谱系统并分析所得数据。FlowJo 用于分析所有流式细胞仪数据。所描述的流式细胞术结果仅使用活细胞群体。通过结合Ca 2+的Indo-1(405/20 nm滤光片)和不含Ca 2+的Indo-1(530/30 nm滤光片)的比例对细胞内钙进行定量。在图4A所示的应答细胞的百分比是从衍生的FlowJo的由模块使用90动力学个百分30米60秒之间的基线测量。对于图2、3B和4B,量化数据分别从Unicorn和FlowJo 导出,并使用Prism显示。图3A和4A是直接从FlowJo 导出的。所有图都是使用Illustrator编译的。


 


笔记


 


通过用铝箔覆盖PhyB 或调暗房间(如果可能),保护PhyB 免受强光照射,例如白光或直射阳光。每当不应该改变PhyB 的构象时,请在绿色安全光下工作,该光应处于PhyB的最低吸光度。白色房间的光线或阳光具有与660 nm照明相似的效果,并将PhyB 转换为ON 状态。
PHYB 是对氧化敏感,因此应保持在由例如脱气溶液和加入0.5还原缓冲毫TCEP,β - 巯基乙醇或二硫苏糖醇。每当实验条件禁止使用的还原剂,期望和监控的下降博士YB 以PIF在几个小时的过程中结合活性。
如Yousefi 等(美国国家科学院,2003年)中所述,连续660 nm的光照可阻止PhyBt 与GFP-PIF S -TCR结合。(2019)。因此,我们可以使用低660 nm的光强度,也可以使用介于两者之间的暗周期的脉冲照明。强度增加的660 nm光照射导致PhyBt 与GFP-PIF S -TCR的结合半衰期缩短。
由于PhyB 的光生物学特性,我们660 nm光源的饱和照明导致80%的分子处于PhyB ON状态,而20%的分子处于PhyB OFF状态。饱和照明与我们的740nm的光源,得到1%PHYB ON和99%PHYB OFF。具有变化的发射光谱的不同光源将导致不同的PhyB ON:OFF比率,有关详细信息,请参见Smith 等。(2016)。
 


菜谱


 


10%NaN 3
将NaN 3 溶于水
室温保存
5 M氢氧化钠
通过将NaOH颗粒缓慢加入水中将其溶解在水中(放热反应)
室温保存
TCEP 50万
将TCEP溶于脱气水中
用5 M NaOH将pH调节至7.4
在-20°C下以1 ml等分试样储存
蛋白质缓冲液
PBS
从0.5 M的储备溶液中添加0.5 mM TCEP
通过0.22 µm过滤器并脱气
不要储存蛋白质缓冲液,但要始终准备新鲜
FACS缓冲区
PBS
1%FBS
0.02%NaN 3 (从10%的库存中稀释)
储存在4°C
刺激培养基
RPMI
1%FBS
10毫米HEPES
储存在4°C
 


致谢


 


我们感谢Signalhaus 机器人设施的Susan Lauw 和Johannes Kaiser 运行机器人平台(INST 39 / 899-1 FUGG)进行钙实验,并感谢Opto Biolabs 为其定制的照明设备。这项工作是由德国资助研究联合会(DFG)在德国的卓越战略,通过EXC294(的BIOS - 中心生物登陆阿灵学),EXC2189(CIBSS- 中心综合生物信号研究,项目编号390939984),SFB854(B19),SCHA976 / 7-1,SCHA976 / 8-1和Project-ID 403222702- SFB 1381(A9)。该协议文件中详细描述的过程来自eLife (Yousefi et al。,2019)中的文章。


 


利益争夺


 


Opto Biolabs 为我们提供了pxONE 设备。


 


参考文献


 


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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Yousefi, O., Hörner, M., Wess, M., Idstein, V., Weber, W. and Schamel, W. W. (2020). Optogenetic Tuning of Ligand Binding to The Human T cell Receptor Using The opto-ligand-TCR System. Bio-protocol 10(5): e3540. DOI: 10.21769/BioProtoc.3540.
  2. Yousefi, O. S., Günther, M., Hörner, M., Chalupsky, J., Wess, M., Brandl, S. M., Smith, R. W., Fleck, C., Kunkel, T., Zurbriggen, M. D., Höfer, T., Weber, W. and Schamel, W. W. A. (2019). Optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor. eLife 8: e42475.
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