Exploiting the RUSH System to Study Lytic Granule Biogenesis in Cytotoxic T Lymphocytes

The Retention Using Selective Hooks (RUSH) system allows for the synchronized release of one or more proteins of interest from a donor endomembrane compartment, usually the endoplasmic reticulum, and the subsequent monitoring of their traffic toward acceptor compartments. Here we describe the RUSH system applied to cytotoxic T cells to characterize the biogenesis of lytic granules, using as a proof-of-concept granzyme B trafficking to this specialized compartment.


Introduction
Cytotoxic T lymphocytes (CTLs) are the T cell effectors specialized for the elimination of cells infected by intracellular pathogens as well as cancerous cells. CTLs exert their cytotoxic function through two well-characterized cytotoxic mechanisms: release of cytotoxic granules (CG) and Fas/FasL mediated apoptosis [1]. The first mechanism involves the release of cytotoxic molecules from CGs via exocytosis at the immunological synapse (IS), a specialized contact area that forms at the interface of the T cell with its cognate target cell [2,3]. Upon TCR-mediated recognition of a cell presenting specific peptide associated with MHCI, CTLs rapidly polarize and reorganize their cytoskeleton to translocate the microtubule-organizing center (MTOC) toward the synaptic interface [4,5]. MTOC docking beneath the IS is a key step in the formation of a lytic synapse as it ensures the microtubule-assisted directional transport of CGs that contain soluble cytolytic proteins, such as granzymes and perforin, that are eventually released into the synaptic cleft [6,7]. The other mechanism by which CTLs exert RUSH system to the study of the biogenesis and intracellular trafficking of the CG components. As a proof-of-concept, we used the RUSH system to synchronize the traffic of granzyme B (GZMB), which is known to exploit the cation-independent mannose-6-phosphate pathway for specific targeting to CGs [12]. As a hook, we used the KDEL sequence, which retains the fused protein in the ER lumen [13]. To verify the correct localization to CGs, which are specialized lysosomes, we co-stained CTLs with the lysosomal marker LAMP-1 at the experiment endpoint. The results provide proof-of-concept that the RUSH system can be exploited to track the traffic of CG-associated proteins in CTLs. This paves the way to dissect the interplay of the pathways that regulate the biogenesis of SCGs and MCGs by synchronizing the trafficking of different CG components in CTLs co-transfected with RUSH constructs expressing the respective reporters. Fig. 1 RUSH system. The RUSH (Retention Using Selective Hooks) system is a two-phase assay. In the initial phase, known also as "retention phase," the reporter protein is anchored in the donor compartment (ER in the example) by the hook due to the streptavidin-SBP (streptavidin-binding peptide) interaction. Addition of biotin in the culture medium introduces the second phase of the assay, known as "release phase." The reporter protein is released from the hook as the result of displacement by biotin and it traffics, together with the associated fluorescent protein, to its acceptor compartment (lysosomes in the example)

Evaluation of RUSH in Live Cells
1. CTLs transfected with RUSH-GZMB construct.

Generation of the RUSH-GZMB Construct
PCR amplification of GZMB is performed using as template a GZMB-mCherry home-made vector. 5 μL of the PCR product is run on an agarose gel (see Note 8) to verify the quality of PCR amplification. The remaining part is purified using NucleoSpin Gel and PCR Clean-up Kit and quantified using QIAxpert system (see Note 9).

Insert and Vector Restriction Enzyme Digestion
Purified GZMB PCR product obtained in paragraph 3.1.1 and Addgene Plasmid #65279 are digested using the following protocol (see Note 10): Add H 2 0 to 60 μL Digestion protocol: 30 min at 37°C and 5 min at 80°C The digested products are loaded onto agarose gels, the DNA fragments are excised from the gel and purified using NucleoSpin Gel and PCR Clean-up Kit and quantified using QIAxpert system. Incubate the reaction at room temperature for 3 h, or 4°C overnight, and inactivate the reaction by heating at 70°C for 10 min.
The ligation reaction obtained is transformed in E. coli DH5α Competent Cells following this procedure: 1. Thaw on ice one tube of DH5α cells.

Plasmid DNA Preparation and Colony Screening
Bacterial cells obtained from single colonies are used to extract the plasmid using the NucleoSpin Plasmid Mini kit for plasmid DNA (Macherey-Nagel or similar). Plasmid DNA obtained (~200-300 ng) is digested with restriction enzymes as described in Subheading 3.1.2 and loaded onto agarose gels to verify the quality of ligated DNA. Plasmid DNA containing the correct insert is selected and a Midiprep is prepared from it using the Nucleo-Bond Xtra Midi Plus EF, Midi kit for endotoxin-free plasmid DNA (Macherey-Nagel or similar). 3. Activate and expand resting CD8 + T cells (day 0) at a cell density of 0.5-2.5 × 10 6 cells/mL in complete R10 medium with 50 U/mL rIL-2 (see Note 15).

Resuspend the Dynabeads
Human T-activator CD3/CD28 in the vial, transfer the desired volume (12.5 μL of beads per 1 × 10 6 of CD8 + T cells) to a tube, add at least 1 mL of PBS 0.1% (v/v) BCS, and mix. Place the tube on a magnet for 1 min and discard the supernatant. Remove the tube from the magnet and resuspend the washed Dynabeads in the same volume of complete R10 medium as the initial volume of Dynabeads taken from the vial.
6. Seed the cell/bead suspension at a cell density of 1 × 10 6 cells/ mL into a flat-bottom 12-well cell culture plate (2 mL/well) and incubate in a humidified CO 2 incubator at 37°C (see Note 16). 48 h after activation remove the beads (day 2) (see Note 17) and expand the cells in complete R10 medium with 50 U/ mL rIL-2 for further 3 days (day 5). 7. Split the cells back to a density of 1×10 6 cells/mL in complete R10 medium with 30 U/mL rIL-2 the day before nucleofection (Fig. 2).

CTL Nucleofection with the RUSH-GzmB Construct
At day 6, count the CTLs obtained and determine cell density. CTL nucleofection with the RUSH-GZMB construct is performed using the Amaxa T cell nucleofector kit, with the following protocol:   10. The next day wash slides (as described in step 5).
11. Add 15 μL/well of a mix of AlexaFluor secondary antibodies, diluted 1:80 each in PBS and keep the slide in a humidified chamber at RT for 45 min (see Note 24).
13. Add 90% (v/v) glycerol in PBS, one drop/well (see Note 25), and then overlay the coverslip, remove the excess of slide mounting solution by very carefully pushing onto the coverslip, then fix it using conventional nail polish.
14. Store the slides at 4°C in the dark.
15. Analyze fluorescence on a confocal microscope to determine the localization of the protein of interest fused to the fluorescent tag. In our setting, where the hook is specific for the ER, the protein of interest should be localized in the ER in the absence of biotin (Fig. 3) and reach its destination compartment when biotin is added to the medium, depending on the kinetics of trafficking of the protein itself (Fig. 4). An example of the results for CTLs transfected with the Str-KDEL_GZMB-SBP-mCherry RUSH constructs is shown in Figs. 3 and 4. 2. LB agar plates with antibiotic selection are obtained as described for the LB liquid medium with the 15 g bacteriological agar per 1 L of water. Mix well by inverting the bottle several times until powder is dissolved and sterilize by autoclaving. Following autoclaving (while media is still liquid but cool enough to safely handle the bottle), add the desired  11. Insert:vector ratio is calculated on the base of insert and vector length (kb) and mass. The ideal ratio is usually 3:1, because a larger amount of insert increases the chances of successful cloning reaction. 13. Plates should be removed from 4°C at least 1 h before use and placed in 30°C incubator.
14. Plating two different volumes of bacterial culture is recommended to ensure that at least one plate will have well-spaced colonies.
15. Split the cells when cell density exceeds 2.5 × 10 6 cells/mL or when the medium turns yellow.
16. Examine cell culture daily, noting changes in cell size and shape.
17. Upon activation, some cells will bind strongly to the beads. Resuspend and transfer the bead/cell suspension to a suitable tube by thoroughly pipetting before cell separation from the beads on a magnet.
18. Avoid storing the cell suspension longer than 20 min, as this reduces cell viability and gene transfer efficiency.
19. Sample must cover the bottom of the cuvette without air bubbles.
20. Use the supplied pipettes and avoid repeated aspiration of the sample.
22. Do not completely dry the wells to avoid cell disruption.
23. The use of anti-RFP antibody in immunofluorescence experiments performed on cells transfected with Str-KDEL_TNF-SBP-mCherry construct permits to amplify the mCherry signal, if required.
24. The fluorescently labelled secondary Ab must be kept safe from the light. For this reason, prepare dilutions and perform incubations in the dark.
25. Dispense slide mounting medium dropwise onto the slide wells using a Pasteur pipette. with LAMP-1 were obtained using Manders' coefficient (mean ± SD; 10 cells/sample). The results show that before biotin addition GZMB has a low co-localization with lytic granules (marked by LAMP-1), while after biotin addition the co-localization increases in a time-dependent fashion, indicating that after release from the ER GZMB is correctly transported to its destination compartment