Multiplex T-cell Stimulation Assay Utilizing a T-cell Activation Reporter-based Detection System.

Immune tolerance and response are both largely driven by the interactions between the major histocompatibility complex (MHC) expressed by antigen presenting cells (APCs), T-cell receptors (TCRs) on T-cells, and their cognate antigens. Disordered interactions cause the pathogenesis of autoimmune diseases such as type 1 diabetes. Therefore, the identification of antigenic epitopes of autoreactive T-cells leads to important advances in therapeutics and biomarkers. Next-generation sequencing methods allow for the rapid identification of thousands of TCR clonotypes from single T-cells, and thus there is a need to determine cognate antigens for identified TCRs. This protocol describes a reporter system of T-cell activation where the fluorescent reporter protein ZsGreen-1 is driven by nuclear factor of activated T-cells (NFAT) signaling and read by flow cytometry. Reporter T-cells also constitutively express additional pairs of fluorescent proteins as identifiers, allowing for multiplexing of up to eight different reporter T-cell lines simultaneously, each expressing a different TCR of interest and distinguishable by flow cytometry. Once TCR expression cell lines are made they can be used indefinitely for making new T-cell lines with just one transduction step. This multiplexing system permits screening numbers of TCR-antigen interactions that would otherwise be impractical, can be used in a variety of contexts (i.e., screening individual antigens or antigen pools), and can be applied to study any T-cell-MHC-antigen trimolecular interaction.

while retaining response to cognate antigens.
Flow cytometric methods, in which a fluorescent reporter gene is driven by response-specific transcription factors such as nuclear factor of activated T-cells (NFAT), sensitively detect T-cell activation ( Figure 1) and can distinguish each cell population by marking with fluorescent proteins (FPs), allowing for the simultaneous evaluation of multiple cell populations. This multiplexing approach reduces the labor and reagents needed for detection of response to antigen when large numbers of TCRs are to be analyzed . Here we describe an NFAT-driven fluorescent reporter system and T-cell multiplexing strategy for large-scale screening of TCR and peptide-MHC complex interactions, to surmount this limiting step in determination of TCR antigen specificity.

Overview of the Procedure
The workflow for this protocol is illustrated in Figure 2. In the first step, TCR expression cell lines are generated using vectors made as described in Steps A1 to A4. Transfection and transduction are used to introduce the vectors ( Figure 1) into 5KC α-β-hybridoma T-cells to generate TCR expression cell lines having all components of the reporter system (along with eight distinct fluorescent identifiers) except TCR α and β chains (Steps B1 to B2c). These TCR expression cell lines can be expanded and frozen, and then used repeatedly for multiple rounds of TCR reporter cell line generation. In the second step, vectors with TCR α and β chains are generated (Step A5) and transduced into the TCR expression cell lines, resulting in functional reporter T-cells (Step B2d). In step 3, APC cell lines are generated expressing MHC genes of biological relevance, depending on the origin of TCRs being analyzed.
Step A6 describes the generation of MHC expression vectors, and vectors are then transduced into APC cell lines (Step B3). In step 4, the multiplex stimulation assay is conducted by combining 8 different FP-identified T-cell reporter lines and co-culturing with APCs and antigens (Steps C1-C4). Finally, flow cytometry is used to measure positively stimulated cells by induction of the reporter color ZsGreen-1 (Step C5). Copyright Figure 2. Procedure workflow. The protocol proceeds in four major steps. The first step generates TCR expression cells lines that are then used in step 2 to make TCR reporter cell lines.
Step 3, generating APC cell lines, can be done concurrently with steps 1 and 2. In step 4, the multiplex stimulation assay is performed, using the TCR reporter lines co-cultured with APC cells and antigens. Flow cytometry is used to measure ZsGreen-1 expression in positively stimulated cells.
A. Vector Description and Construction 1. Refer to Table 1 for a list of all vectors mentioned in Section A, which are available from Addgene. Table 2 lists all primers used in Section A for vector construction and verification by PCR and sequencing. Copyright   2. hCD8-8xNFAT reporter vector. This vector is to introduce the T-cell activation reporter gene as well as TCR co-receptor genes such as human CD8. As shown in Figure 3, the 8xNFAT-ZsG-hCD8 reporter vector is a replication-incompetent and self-inactivating retroviral vector with the 8xNFAT-ZsG reporter construct (8 repeats of NFAT binding sites in a row, followed by a TATA box and the ZsGreen-1 gene) inserted into the site behind truncated gag a. Optional step: Replace the hCD8 genes with hCD4 genes or another TCR co-receptor.
Refer to the NCBI nucleotide website (https://www.ncbi.nlm.nih.gov/nucleotide/) to obtain the coding sequence for hCD4 or the desired genes.
i. Design a nucleotide sequence for the DNA fragment to be inserted into the vector.  2) Warm LB-ampicillin plates (see Recipes) in 37 °C incubator.
3) Plate the entire transformation mixture onto pre-warmed LB-ampicillin plates and incubate overnight at 37 °C.
x. Grow mini-cultures of individual colonies and perform PCR to verify presence of the DNA fragment insert. Sequences for all primers used for PCR are given in Table 2. 2) Cover the mini-cultures with a foil plate seal and incubate for 4 h at 37 °C, shaking at 250 rpm.
3) Prepare a PCR master mix with the component ratios given in Table 3. Mini-culture 14 www.bio-protocol.org/e3883       Table 3, with the following primers: ix. The F Primer (CD3-Seq) and the R Primer (SalI-Seq) locate before the MfeI recognition site and behind the HpaI recognition site in the 7,263 bp vector backbone fragment, respectively ( Figure 4). Carry out PCR using the conditions given in Table 4, except using an annealing temperature of 50 °C with an elongation time of 2.5 min.
x. Visualize PCR products by separation on a 1% agarose gel with 0.5 μg/μl ethidium bromide. Correct DNA fragment insertion will result in a PCR product equal to the length of the FP gene plus 746 bp.
xi. Prepare vector for transfection. Proceed to large-scale culture plasmid preparation from the mini-culture of one colony having the correct-sized PCR product as confirmed by mini-culture PCR as in Step A2a.xi.
xii. Check the vector for correct sequence and alignment of inserted DNA fragment by submitting samples for Sanger sequencing with the primers shown in Table 2 for 3. Fluorescent Protein (FP) Vector. As seen in Figure 5, which shows FP mCherry www.bio-protocol.org/e3883 [pMSCVII-mChe (pMSCV-mChe), Figure 5A] as an example, the FP genes are sub-cloned into a non-replicating MSCV-based retroviral vector (pMSCVII, Figure 5B)  www.bio-protocol.org/e3883  Table 3, with the following primers: The F Primer (5pMIG) and the R Primer (3LTR-2) located before the EcoRI recognition site and behind the XhoI recognition site in pMSCVII, respectively ( Figure 5).
i. Carry out PCR using the conditions given in Table 4, except using an annealing temperature of 54 °C and an elongation time of 1.5 min.
j. Visualize PCR products by separation on a 2% agarose gel with 0.5 μg/μl ethidium bromide.
Correct DNA fragment insertion will result in a PCR product equal to the length of the FP gene plus 226 bp.
k. Prepare vector for transfection. Proceed to large-scale culture plasmid preparation from the mini-culture of one colony having the correct-sized PCR product from mini-culture PCR as in Step A2a.xi.
l. Check the vector for correct sequence and alignment of inserted DNA fragment by submitting samples for Sanger sequencing with the primers shown in Table 2 for pMSCVII.     Table 5.
ii. Incubate reactions at 50 °C for 1.5 h, and then hold at 4 °C.
iii. After incubation, reactions can be stored at 4 °C overnight and used to transform NEB   Table 3, with the following primers: The F Primer (5MAC3) and the R Primer (3TRBC) locate within DNA Fragment 2 Cα-P2A and behind the BglII recognition site in MBC1(or 2)-null, respectively.
g. Carry out PCR according to the conditions given in Table 4, except using an annealing temperature of 60 °C and an elongation time of 1.0 min.
h. Visualize PCR products by separation on a 2% agarose gel with 0.5 μg/μl ethidium bromide.
Correct DNA fragment insertion will result in a PCR product 600 ± 20 bp for α and 680 ± 20 bp for β.  Figure 10, MHC gene cassettes are inserted into the multi-cloning site (MCS) of a non-replicating lentiviral vector containing a ubiquitous chromatin opening element (UCOE)-preceded spleen focus forming virus (SFFV) promoter (pUS, Figure   10A). Figure 10B illustrates MHC I gene vector A2_uSFFV, which is used in the example stimulation assay in part B.  e. Set up a Gibson assembly reaction according to the reaction ratios shown in Tables 6 and   7.   Table 3, with the following primers: Cas9-S16 (F Primer): 5'-AAGAGCTCACAACCCCTCAC-3' Cas9-S14 (R Primer): 5'-CACATAGCGTAAAAGGAGC-3' The F Primer (Cas9-S16) and the R Primer (Cas9-S14) locate within before and behind the MCS of the pUS vector, respectively.
j. Carry out PCR using the conditions given in Table 4, except using an annealing temperature of 50 °C and an elongation time of 2.5 min.
k. Visualize PCR products by separation on a 1% agarose gel with 0.5 μg/μl ethidium bromide.  Tables 8 and 9 give an overview of the cells and reagents used in both retroviral and lentiviral transfections and transductions. Figure 11         ii. Transduction. Note that Table 9 lists the conditions used in retroviral transductions.

4) Collect Phoenix cells by removing with
1) Thaw 5KC α-β-cells on the same day that Phoenix cells are plated on PDL plates.
Place a cryovial containing 1 x 10 6 cells in 37 °C water bath until the last bit of ice is gone. Carefully transfer cells into a 15-ml centrifuge tube containing 10 to 12 ml 5KC medium (Recipe 2) using wide bore pipette tip.
2) Centrifuge cells at 315 rcf at room temperature for 3 min.
3) Remove medium above the cell pellet, gently resuspend cells, and transfer to a T25 flask containing a total of 10 ml of medium. Grow in CO2 incubator overnight. (iv) Remove 5 ml of medium from each transfected Phoenix cell plate using a disposable syringe and filter through a 0.45 μm syringe filter into a 14-ml round-bottom culture tube. This is now referred to as the retroviral supernatant.
To continue producing retroviruses for the second day of transduction, add 5 ml of prewarmed Phoenix medium back to each Phoenix cell plate and return to incubator.
(v) Add 1 ml of 8xNFAT-ZsG-hCD8 and 1 ml of CD3-AM or CD3-LO retroviral supernatants into 5KC α-β-cells in each transduction well on the 12-well plate made in step (ii). An example plate map is shown in Figure 13.   Note: Reagents for retroviral transfections can be found in Table 8.
iii. Transduction Step B2b.ii to perform transduction.
Note: Reagents for retroviral transductions can be found in Table 9.
2) Repeat transduction the following day.
3) Expand transduced 5KC α-β-cells for 3 days, sub-culturing each day as described in Step B2b.iii.   Note: Reagents for retroviral transfections can be found in Table 8.

4) After 3 days, collect all cells by centrifugation
2) Transduce the 8 color-coded reporter cell lines with TCR vectors according to the plate map shown in Figure 17. Follow steps in Step B2b.ii, adding 2 ml of retroviral supernatant per well.
Note: Reagents for retroviral transductions can be found in Table 9.
3) Repeat transduction the following day.
4) Expand transduced 5KC cells for 3 days, sub-culturing each day. except using a lentiviral packaging system instead of a retroviral system (Tables 8 and 9).

5) Collect cells and enrich CD3+ cells using a Miltenyi MACS mouse
The MHC vector A2_uSFFV is used as an example in this protocol for the generation of K562 cells expressing HLA-A*02:01.
Subculture daily according to manufacturer's instructions.
2) Prepare one PDL-treated plate of 293FT cells for each MHC expression vector and transfect using the lentiviral system, reagents for which can be found in Table 8.
Note: The transfection procedure for the lentiviral system follows the steps described in Step B2b.i, except using cell lines and packaging vectors as outlined in 2) Prepare a 6-well transduction plate seeded with 5 x 10 5 K562 cells in each well according to the parameters for lentiviral transduction given in Table 9. Prepare one well for each transduction.
3) Prepare lentiviral supernatant following the steps described for retroviral transduction, add 2 ml of lentiviral supernatant to each well of transduction plate, and perform one day of transduction according to parameters given in Table 9.  Figure 18 shows the schedule for a stimulation assay experiment and a schematic for plate set up. Copyright    i. Set compensation for all fluorescent colors used before running stimulation plate.
Each fluorescent protein appears in the channel as in Table 10. Resuspend cells in 150 μl Phenol red-free RPMI using a multichannel pipettor.
iii. Acquire data for 75,000 cells per well (sufficient to acquire at least 3,000 5KC-TCR cells expressing each color combination) using a flow cytometer equipped with a plate-loading function.
1) Flow rate can be set between 100 and 120 μl/min on Beckman-Coulter Cytoflex.

Data analysis
A. Import data into FlowJo for analysis.
B. Gate cells according to the gating strategy shown in Figure 19.
Determine percent positivity of ZsGreen-1 from each well and import into an Excel file. Cells that exhibit higher intensity signals in the FITC channel than signals of the majority (i.e., >97-99%) of non-activated control cells are determined as positive for ZsGreen-1 expression. Copyright   3. The positive control is used to confirm that the TCR/CD3 complex is expressed and functional.
www.bio-protocol.org/e3883 6. For the construction of TCR vectors, backbone vector choice is determined by the TRBJ gene.
7. When freezing and thawing cells, we recommend using wide-bore P1000 pipette tips to minimize mechanical stress on cells (see Equipment list).
a. After thawing, save a small portion of cells (<100 μl), make a 1:1 dilution with Trypan Blue, and count cells on a Countess II Automated cell counter. Divide the number of live cells by total number of cells to attain cell viability.
Expected viability is ≥ 90% b. Phoenix-ECO cells often require 1-2 days to achieve normal cell growth rate and conditions after thawing. This can be assisted by: i. Using an earlier passage number of cells.
ii. Increase FBS in thawing media to 20%.
iii. Treating culture dish with PDL prior to thawing to assist cells in adhering.
8. 5KC α-β-and K562 culture conditions should be optimized prior to starting transfection and transduction procedures. Optimization should include: a. Manually checking and calibrating CO2 levels of 37 °C incubator to 5% CO2 to prevent acidification of medium.
b. Testing multiple (2 or 3) lots of FBS for effect on cell growth rate and condition. Atlanta Biologicals is a recommended source of FBS for 5KC cells.
c. Determining sub-culturing conditions that maintain healthy 5KC α-β-growth rate and condition. 5KC α-β-cells prefer sparse conditions rather than dense. In general, this requires splitting cell cultures every day.
i. When cells are seeded at 5 x 10 4 cells/ml, they achieve a doubling rate of ≤12 h.
ii. Cell condition is assessed using an inverted light microscope. Cells should appear bright with smooth edges and a roundish shape with occasional small blebs or points.
d. K562 growth conditions should be optimized based on manufacturer's instructions. In Copyright  general, they grow more slowly than 5KC α-β-cells and are therefore seeded at a higher density. They appear larger and more rounded than 5KC cells when visualized with an inverted light microscope. 9. The CD3-containing vectors (CD3-AM and CD3-LO) contain fluorescent protein genes because CD3 will not be expressed by 5KC α-β-cells before TCR genes have been added, and therefore fluorescence is used as a marker for cells that are transduced with the vector. b. The cells generated in this step should be handled in the best manner possible and sufficiently expanded to allow many (> 16) vials to be frozen for future use. Avoid prolonged passaging of these cell lines before using to generate 5KC-TCR cell lines or freezing down additional vials.
11. When TCR is not expressed well: a. Some primary T-cells contain two α chains, often one functional and one non-functional.
When two TCRs of a set share the same β chains with two α chains, the non-functional TCR may not be reconstituted.
b. Some human V-α and V-β regions may be incompatible with the mouse signal peptide regions to be cleaved correctly. Replacing the mouse signal peptide region with an intrinsic human signal peptide region sequence may improve TCR expression.
c. We do not use codon-optimized nucleotides to express chimeric TCR genes in murine T-hybridoma cells, 5KC α-β-cells, since it is our experience that natural nucleotide sequences typically result in higher TCR/CD3 expression than the codon-optimized sequences. However, codon-optimization may occasionally improve TCR/CD3 expression for some TCRs.
12. Both 5KC and K562 cells must be treated as if they are shedding virus particles (non-replicating) for three complete media exchanges after transduction. Carry out all cell handling from transfection up to this point in BS level 2 conditions. 13. PBS is added to empty wells on culture plates to prevent excessive evaporation of media in dry climates. In humid climates this may not be necessary.
14. Various types of cells other than K562-APCs can be used as APCs in stimulation assays.
Examples include autologous B cell lines transformed with Epstein-Barr virus, irradiated autologous peripheral blood mononuclear cells, and dendritic cells isolated/propagated/differentiated from autologous peripheral blood cells. The APC concentration needs to be optimized for each type of APCs.