Flow cytometric analysis of CD4+ T cell reactivation following anti-PD1 immunotherapy in a transgenic mouse model

Summary This protocol uses the Tg4 Nr4a3-Tocky mouse model to recalibrate T cell activation thresholds and reveals the role that immune checkpoints play in controlling T cell activation. The example approach here uses flow cytometry to characterize quantitative and qualitative changes in splenic CD4+ T cells reactivated in the presence of anti-PD1 immunotherapy. The protocol is optimized for studying anti-PD1 pathway blockade only. The protocol is not compatible with cellular fixation, and T cells should be analyzed immediately after staining. For complete details on the use and execution of this protocol, please refer to Elliot et al. (2021).

Prepare Tg4 Tiger Nr4a3-Tocky mouse line The protocol uses the F1 generation from the breeding of Nr4a3-Tocky mice (C57BL6, I-A b ) crossed with the Tg4 Tiger (Il10-GFP) line, which is on the B10.PL background (Burton et al., 2014). Nr4a3-Tocky mice are NFAT-responsive distal TCR signaling reporter mice that express fluorescent timer (Timer) protein (Subach et al., 2009) under the control of Nr4a3 regulatory elements (Jennings et al., 2020). The Tg4 TCR transgenic is specific for a peptide derived from myelin basic protein (MBP) presented by the I-A U MHC Class II molecule (Figure 1). We maintain colonies of Nr4a3-Tocky mice as homozygous BAC transgenics . Similarly, Tg4 Il10-GFP are bred to homozygosity. This set up means no genotyping is required for the F1 generation as all will be ''hemizygous'' for transgenes. The protocol does not necessitate the presence of the Il10-GFP transgene, and Tg4 mice can be crossed with Nr4a3-Tocky mice for use with the protocol. The protocol depends on the expression of Timer protein to capture the re-activation of T cells within the 4 h time frame. In theory, any TCR transgenic systems could be mated to the Nr4a3-Tocky line (e.g., OTII), although these have not been experimentally tested within our laboratory.
4. Purchase 10 mg-50 mg of MBP (custom product from GL Biochem Shanghai, sequence Ac-AS-QYRPSQR, >90% purity). 5. Upon arrival, reconstitute the powder in sterile dd H 2 0 to a stock concentration of 4 mg/mL and freeze it in aliquots until use. We typically prepare 200 mL aliquots and freeze at À20 C.
CRITICAL: Avoid freeze/thaw cycles and always use fresh aliquots. Always keep peptides on ice when transporting.

Prepare anti-PD1
Anti-PD1 preparation: 6. Purchase in vivo grade anti-PD1, clone 29F.1A12 (available from BioLegend or BioXcell) and isotype control (rat IgG2A). We use rat IgG2a (clone MAC219 (Forsyth et al., 2000)) . 7. Anti-PD1 and the isotype controls are stored in the fridge (4 C) until use. The panel design has been optimized to minimize interference with the Timer-Blue channel (excitation 405 nm, detection 450/50 nm filters). We have previously shown that at physiological expression levels, GFP (excitation 488 nm detection 530/30 nm filers), Timer Blue and Timer Red (excitation 561 nm, detection 610/20 nm filters) show negligible compensation requirements . A key compensation consideration is the overlap of PE-Cy7 and PE-Texas Red (Timer-Red) channels. We strongly recommend that for PE-Cy7 compensation, the same antibody is used for single color controls.  For further details regarding compensation matrix considerations and fluorochrome compatibility with Nr4a3-Timer Red, Nr4a3-Timer Blue and GFP please refer to   CRITICAL: A bolus shape should form under the skin if injection is subcutaneous. If significant amounts of peptide/PBS solution leaks after injection (>5-10 mL) then that mouse should be excluded from the experiment.

Spleen harvest and generation of splenocyte suspensions
Timing: 1-2 h 4. After 4 h peptide rechallenge, euthanize mice, and process spleen for flow cytometric staining a. Mice are euthanized and the whole spleen is removed intact from the Tg4 Nr4a3-Tocky mice. Ensure complete detachment of the pancreas and place it into a 1.5 mL Eppendorf tube containing 0.5 mL of 2% FBS/ PBS (v/v). b. Into a sterile 5 mL petri dish, place a 70 mm cell strainer and add the spleen and 1 mL of 2% FBS PBS buffer. (Alternatively, a strainer can be placed in a 50 mL Falcon tube for processing). c. Using the syringe plunger from a 5 mL syringe, gently force the spleen through the cell strainer. d. Using a P1000 pipette add 1 mL 2% FBS PBS buffer to wash the strainer. e. Filter the splenocyte solution back through the strainer by taking from suspension in the petri dish and pipetting back onto the strainer. i) Repeat this 8-10 times using P1000 pipette until suspension is homogenous. f. Transfer the splenocyte suspension from each mouse into individual 15 mL Falcon tubes. g. Wash strainer with 1 mL 2 % FBS PBS and transfer this to the 15 mL Falcon tube. h. Centrifuge Falcon tube at 500 g for 5 min at 15 C-25 C. i. Decant supernatant and proceed to perform red blood cell lysis by resuspending the cell pellet in 1 mL of RBC lysis buffer. j. Incubate for 2 min on ice. k. Top up falcon tube to 10 mL with cold 2% FBS PBS and centrifuge at 500 g for 5 min at 4 C. l. Decant supernatant and resuspend splenocytes in 2 mL ice cold 2% FBS PBS.
i. Remove with a P1000 pipette any cell clumps that may have formed. m. Transfer 100 mL of splenocyte suspension (typically 2-5M splenocytes) to a 96 well U bottom plate for staining. Alternatively, cells can be stained in 5 mL FACS tubes. CRITICAL: Do not fix cells as this will result in loss of Nr4a3-Timer Blue fluorescence. Due to slow maturation of Timer protein from Blue to Red it is advised that data are acquired soon after completion of staining. Troubleshooting 5

EXPECTED OUTCOMES
Treatment with anti-PD1 will increase the proportion of T cells that re-activate in response to re-challenge with the 8-mg dose of peptide in comparison to isotype treated mice. These cells are defined as the percentage of CD4 + T cells that are Nr4a3-Timer Blue + Red + (Figure 2). Furthermore, analysis of CD4 + Nr4a3-Timer Blue + Red + T cells will reveal that the median level of Nr4a3-Timer Blue and OX40 will be higher in responder T cells in the anti-PD1 treated group compared to isotype treated mice (Elliot et al., 2021). In addition, Il10-GFP expression will be found within the Nr4a3-Timer Red fraction (Figure 3).

QUANTIFICATION AND STATISTICAL ANALYSIS
Flow cytometry gates to determine responder populations can be set by including a mouse that receives the primary immunization (80 mg [4Y] MBP) but only PBS on the secondary restimulation. Most T cells, after 24 h immunization with 80 mg [4Y] MBP, are Nr4a3-Timer Red + Blue - (Figure 4). When a second immunization with 8 mg [4Y] MBP is performed a clear Blue + Red + population is observable. These are the ''responder'' cells. Gating on this population will allow a comparison of the phenotype of responder cells between treatment groups. As part of a quality control step, we include a fluorochrome conjugated anti-PD1 antibody of the same clone as the blocking antibody administered in vivo. This allows confirmation of successful i.p. injection and that PD1 is blocked ( Figure 5). Where significant PD1 staining remains, this indicates inadequate blocking of PD1, and such mice should be excluded from further analysis.

LIMITATIONS
The protocol here is optimized for anti-PD1 immunotherapy but has been adapted for other immune checkpoints such as Lag3; however, for other receptors confirmation that the receptor and its ligand are expressed in the splenic environment would be required. Due to the mixed mouse background, generation of gene knockouts is not straightforward, and would require backcrossing onto the B10.PL background.
The protocol is specific for CD4 + T cells, but theoretically could be adapted to CD8 + T cells using a CD8 + TCR transgenic. However, for all analyses of responder T cells, the protocol is not compatible with fixation and intranuclear staining and samples need to be analyzed soon after completion of flow cytometric staining.

Potential solution
High variability is often driven by inadequate randomization. We typically use mice 5-6 weeks of age with weights 18-24 g. We ensure mice are randomized based on sex and age, as both influence weight. As multiple injections are given over time, large variance in mice weight can give rise to wider ranges in responses. One way to circumvent this is to administer doses on a mg/ kg basis. We base our 80-mg dose

OPEN ACCESS
on an approximate mouse weight of 20 g. This equates to doses of 4 mg/kg for primary immunization and 0.4 mg/kg for re-challenge. For antibody, 0.5 mg would equate to 25 mg/kg.

Problem 2
Timer-Red channel shows a skewed distribution (step 5).

Potential solution
This is typically a result of over or under compensation of PE-Cy7 and Timer Red (PE-Texas Red channel). In our experience PE-Cy7 tandem dyes exhibit subtle differences in compensation between batches and brands. It is imperative to use the same PE-Cy7 antibody as in the panel (either using beads compensation, or staining of cells expressing the ligand).

Potential solution
You can utilize a ''dump'' channel to remove noise. We recommend using the PerCP-Cy5.5 channel (excited by a 488 nm laser, with 710/50 nm filters) to remove cells showing signal in this channel.

Problem 4
Minimal effect of anti-PD1 on T cell reactivation (step 2c).

Potential solution
There are several potential reasons for this. (1) A key part of the protocol is the minimum 30-min gap between administration of the anti-PD1 antibody and re-challenge with [4Y] MBP. This gap can be extended to increase the time for maximal blockade of the pathway for antibodies with potential different distribution half-lives. (2) It is strongly recommended that receptor blockade is confirmed through counter staining for the marker of interest. In our hands, a non-response is almost always due to a failed i.p. injection as evidenced by high PD1 staining.

Potential solution
This could represent a failed subcutaneous injection of 8 ug [4Y] MBP, which would result in no restimulation of the T cells in vivo. Conversely, this can arise if cells are left overnight in the fridge or fixed, as Timer Blue will undergo slow maturation to the terminal Red form, resulting in loss of Blue signal. It is imperative that the experiment is planned so that flow cytometric analysis can occur soon after completion of staining.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr David Bending, d.a.bending@bham.ac.uk .

Materials availability
Nr4a3-Tocky mice are available under MTA from Dr Masahiro Ono (Imperial College London, UK).

Data and code availability
Data underlying this protocol are available from the lead contact upon reasonable request.

ACKNOWLEDGMENTS
This work is funded by the MRC (MR/V009052/1). The graphical abstract and Figure 1 were created using https://biorender.com.

DECLARATION OF INTERESTS
The authors declare no competing interests.