Materials and Reagents

Eppendorf^® Safe-Lock microcentrifuge 1.5 mL tubes (Sigma-Aldrich, catalog number: T9661)
70 μm Cell Strainer (Vita Scientific, catalog number: NSTF90051)
CytoOne T75 filter cap TC flask (USA Scientific, catalog number: CC7682-4875)
100 mm Dish, Tissue Culture-Treated (VWR, catalog number: 25382-428)
Corning^® 96-well Clear Flat Bottom Polystyrene TC-treated Microplates (Corning, catalog number: 3596)
0.45 µm Sterile Polyethersulfone Syringe Filter (VWR, catalog number: 28145-493)
10 mL BD Luer-Lok^TM Syringe (BD, catalog number: 309605)
Axygen 96 Well Clear V-Bottom 550 µL Polypropylene Deep Well Plate (Axygen, catalog number: P-96-450V-C)
IL10^GFP/Foxp3^RFP dual reporter mouse model (generated as we previously reported in Huang et al., 2017)
Rag^-/-mouse model (Jackson Laboratory, Strain #: 002216)
Note: All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the Louisiana State University (protocol #: 18-047 and 21-039).
MS Columns (Miltenyibiotec, catalog number: 130-042-201)
LS Columns (Miltenyibiotec, catalog number: 130-042-401)
InVivoMAb anti-mouse CD3ϵ [clone: 145-2C11] (Bio X Cell, catalog number: BE0001-1)
InVivoMAb anti-mouse CD28 [clone: 37.51] (Bio X Cell, catalog number: BE0015-1)
Mouse IL-27 (NS0-expressed) Protein (R&D Systems, catalog number: 2799-ML-010)
Retro-X^TM Concentrator (Takara Bio, catalog number: 631456)
Opti-MEM^TM, Reduced Serum Medium (Thermo Fisher Scientific, catalog number: 31985070)
Mirus Bio TransIT^®-293 Transfection Reagent (VWR, catalog number: 10766-968)
Plat-E cells, a retrovirus packaging cell line described in Morita et al. (2000)
Bicistronic retroviral vector (e.g., pGC-IRES-Thy1.1, pMI-IRES-hCD2, pGC-IRES-BFP2. See Acknowledgments for details).
RPMI 1640 Medium (Fisher Scientific, catalog number: 11-875-119)
DMEM (Fisher Scientific, catalog number: 11-965-118)
Fetal Bovine Serum (Thermo Fisher Scientific, catalog number: 10082147)
1 M HEPES solution (Cytiva, catalog number: SH30237.01)
100× Nonessential Amino Acids (Corning, catalog number: 25-025-ci)
100 mM Sodium Pyruvate (Hyclone, catalog number: sh30239.01)
200 mM L-Glutamine (Hyclone, catalog number: SH30034.02)
100× Penicillin/Streptomycin Solution (Hyclone, catalog number: SV30010)
Puromycin dihydrochloride (Sigma-Aldrich, catalog number: P7255)
Blasticidin S.HCl Solution in HEPES buffer 10 mg/mL (A.G. Scientific, catalog number: B-1247-SOL)
Red Blood Cell (RBC) Lysis Buffer (10×) (Tonbo Biosciences, catalog number: TNB-4300)
Anti-biotin Microbeads (Milteny Biotec, catalog number: 130-090-485)
Mitomycin C (CAS 50-07-7) (Santa Cruz Biotechnology, catalog number: SC-3514B)
3% Acetic Acid with Methylene Blue (Stemcell^TM Technologies; catalog number: 07060)
Biotin Anti-human/mouse CD45R (B220) [clone: RA3-6B2] (Tonbo Biosciences, catalog number: 30-0452)
Biotin Anti-CD19 monoclonal antibody [clone: HIB19] (Thermo Fisher Scientific, catalog number: 13-0199-82)
Biotin Anti-CD8α rat monoclonal antibody [clone: 53-6.7] (Biolegend, catalog number: 100704)
Biotin Anti-mouse TER-119 [clone: TER-119] (Tonbo Bioscience, catalog number: 30-5921)
Biotin Anti-CD49b (pan-NK cells) rat monoclonal antibody [clone: DX5] (Biolegend, catalog number: 108904)
Biotin Anti-NK1.1 [clone: PK136] (Tonbo Bioscience, catalog number: 30-5941)
Biotin Anti-TCRγδ [clone: GL 3] (eBioscience, catalog number: 13-5711-85)
Biotin Anti-Ly-6G/Ly-6C (Gr-1) rat monoclonal antibody [clone: RB6-8C5] (Biolegend, catalog number: 108404)
Biotin Anti-mouse CD122 (IL-2Rβ) [clone: 5H4] (Biolegend, catalog number: 105904)
Biotin Anti-CD25 rat monoclonal antibody [clone: PC61] (Biolegend, catalog number: 102004)
Biotin Anti-CD44 rat monoclonal antibody [clone: IM7] (Biolegend, catalog number: 103003)
Biotin Anti-CD11b rat monoclonal antibody [clone: M1/70] (Biolegend, catalog number: 101204)
Biotin anti-CD11c monoclonal antibody [clone: 3.9] (Thermo Fisher Scientific, catalog number: 13-0116-82)
Biotin Anti-mouse F4/80 [clone: BM8] (Biolegend, catalog number: 123106)
Ghost Dye^TM Violet 510 (Tonbo Biosciences, catalog number: 13-0870-T100)
FITC Anti-mouse TCRβ [clone: H57-597] (Tonbo, catalog number: 35-5961)
Phycoerythrin (PE) Anti-mouse CD25 monoclonal antibody [clone: PC61.5] (Tonbo Biosciences, catalog number: 50-0251)
Allophycocyanin (APC) Anti-CD4 rat monoclonal antibody [clone: GK1.5] (Biolegend, catalog number: 100412)
PE-Cy7 Anti-CD62L rat monoclonal antibody [clone: MEL-14] (Tonbo Bioscience, catalog number: 60-0621-U100)
Pacific Blue^TM Anti-human CD2 (hCD2) antibody [clone:TS1/8] (Biolegend, catalog number: 309216)
PE Anti-rat CD90/mouse CD90.1 (Thy-1.1) antibody [clone: OX-7] (Biolegend, catalog number: 202523)
Full DMEM (see Recipes)
Full DMEM + Puromycin/Blasticidin (see Recipes)
Full RPMI (see Recipes)
1× RBC Lysis Buffer (see Recipes)
Primary biotin antibodies master mix (see Recipes)
Magnetic anti-biotin microbeads + sorting antibody master mix (see Recipes)

Equipment

LSR Fortessa X-20 (BD Biosciences)
FACS Aria II Cell Sorter (BD Biosciences)
Avanti J-15R Centrifuge (Beckman Coulter)
Eppendorf Centrifuge 5420 (Eppendorf AG)
MACS^® MultiStand (Miltenyi Biotec, catalog number: 130-042-303)
Forma^TM Series 3 Water Jacketed CO₂ Incubator (Thermo Fisher, catalog number: 4110)

Software

FlowJo (FlowJo, LLC)
FACS DIVA software version 9.0 (BD Biosciences)
GraphPad Prism (GraphPad Software)

Procedure

Retrovirus Production
Note: All handling of retroviral production and transduction occurred under a BSL-2 hood. RV produced from Plat-E are replication incompetent and ecotropic. However, many commonly used genes of interest have been identified as oncogenes, e.g., HRas^G12V (Huang et al., 2017).
1. Maintain Plat-E cells in a T75 flask with full DMEM + Puromycin/Blasticidin (see Recipes).
2. Seed 4 × 10⁶ Plat-E cells into a 100 mm dish with full DMEM media without Puromycin/Blasticidin.
3. Transfect Plat-E cells at ~70%–80% confluent, normally ~18–24 h after seeding.
4. Warm OPTI-MEM and TransIT^®-293 to room temperature.
5. Pipette 1 mL of OPTI-MEM into a polypropylene 1.5 mL Eppendorf tube.
6. Add 10–15 μg bicistronic retroviral vector plasmid DNA to OPTI-MEM. Gently pipette to mix.
  Note: Adjust total plasmid for optimal transfection efficiency. In our experience, 10–15 μg is appropriate for most plasmids.
7. Before use, vortex TransIT^®-293 for 0.5 seconds. Pipette 30–45 μL TransIT^®-293 to the OPTI-MEM + plasmid mixture. Gently pipette to mix.
  Note: A ratio of 1 μg plasmid DNA to 3 μL TransIT^®-293 works well for most plasmids. Adjust total amount of plasmid DNA and ratio DNA:TransIT^®-293 reagent to optimize.
8. Incubate transfection mixture at room temperature for 20 min.
9. Add transfection mixture dropwise to cells, evenly distributed across the plate. Rock gently back and forth to spread evenly.
10. Return cells to incubator.
11. At 24 h post-transfection, collect culture media, and replace with fresh prewarmed full DMEM media.
12. Centrifuge the collected media at 600 × g and 4 °C for 5 min, then pass through a 0.45 μm filter into a clean 50 mL tube. Store filtered media at 4 °C.
13. Repeat steps 11 and 12 at 48- and 60-h post transfection, and combine filtered media into a single 50 mL tube.
14. Add one volume of Retro-X concentrator per three volumes of filtered media.
15. Gently invert tube to mix.
16. Incubate at 4 °C overnight.
17. Centrifuge at 1,500 × g and 4 °C for 45 min.
18. Discard supernatant. Resuspend pellet in 1/10^th of the original volume, in full RPMI media.
19. Use concentrated RV media immediately, or store at -80 °C in single use aliquots. Under these conditions, RV may be stored up to 2 years after production.
1. Isolation of Naïve CD4⁺ T cells
1. Collect spleen and lymph nodes (superficial cervical, brachial, and inguinal are easily accessible) from IL10^GFP/Foxp3^RFP dual reporter mouse model.
2. Homogenize tissues through a 70 μm cell strainer via grinding, and transfer to a 15 mL centrifuge tube.
3. Centrifuge at 600 × g for 5 min.
4. Discard supernatant and resuspend cells in 1 mL of 1× RBC lysis buffer (see Recipe 4). Add RBC Lysis Buffer to 5 mL. Incubate at room temperature for 5 min.
5. Add 2 mL of full RPMI media to neutralize the lysis reaction. Centrifuge at 600 × g for 5 min.
6. Discard supernatant, resuspend in 1 mL of full RPMI media, and transfer to a 1.5 mL tube.
7. Centrifuge at 1,500 × g for 1 min, aspirate supernatant, and resuspend in primary biotin antibody master mix (see Recipe 5), using 200 μL per mouse. Remove irregular clumps if any are present.
8. Incubate on ice for 15 min, shaking every 5 min.
9. During incubation, prepare magnetic anti-biotin microbeads + sorting antibody master mix (see Recipe 6).
10. Add 1 mL of full RPMI media to cells. Centrifuge at 1,500 × g for 1 min, aspirate supernatant, and resuspend in magnetic anti-biotin microbeads + sorting antibody master mix.
11. Incubate cells on ice for 15 min, shaking every 5 min.
12. While cells are incubating, prepare MS or LS magnetic separation columns on the MACS Multistand. Pre-wet columns with full RPMI media at room temperature. Load a sterile collection tube under the magnetic separation column.
  1. MS column holds one spleen. Pre-wet with 0.5 mL of full RPMI media.
  2. LS column holds four spleens. Pre-wet with 2 mL of full RPMI media.
13. Add 1 mL of full RPMI media to cells. Centrifuge at 1,500 × g for 1 min, and aspirate supernatant.
14. Resuspend cells in 550 μL of full RPMI media. Take 500 μL from the top of the tube, and load into magnetic separation columns. Allow media to flow through.
15. To increase yield, add 550 μL of RPMI to the 1.5 mL tube. Take 500 μL from the top of the tube, and load into magnetic columns.
  Allow cells to flow through the column into the sterile collection tube. The naïve T cells are in the flow-through and are ready for sorting (Figure 1A).
  
  Figure 1. Naïve CD4⁺ T Cell Isolation Gating Strategy. Schematic representation of naïve CD4⁺ T cell isolation (A). Representative FACS plots of gating strategy for isolating naïve CD4⁺ T cells by flow sorting for in vitro differentiation (B). Adapted from Huang et al. (2017).
16. Prepare flow sorting collection tubes by adding 1 mL of cold full RPMI media to 15 mL tubes.
17. To isolate naïve CD4⁺ T cells, sort CD44^loCD62L^hiCD25-Foxp3^RFP-TCRβ⁺CD4⁺ cells via FACS Aria II Cell Sorter (BD Biosciences). An example of the gating strategy is presented in Figure 1B (Huang et al., 2017).
18. Centrifuge purified naïve CD4⁺T cells at 600 × g for 5 min. Discard supernatant. Resuspend at 1 × 10⁶ cells/mL in full RPMI media.
1. Tr1 Cell Differentiation
1. To obtain antigen presenting cells (APCs), homogenize spleen of Rag^-/- mouse through a 70 μm strainer into 5 mL of full RPMI media.
2. Add Mitomycin C to a final concentration of 50 μg/mL. Incubate at 37 °C for 30 min.
3. Centrifuge at 600 × g for 5 min. Discard supernatant.
4. Resuspend cells in 1 mL of full RPMI media. Transfer to a 1.5 mL tube.
5. Wash cells by centrifuging at 1,500 × g for 1 min. Aspirate supernatant. Resuspend in 1 mL of full media. Repeat wash step a total of four times.
6. Aliquot 10 μL of cells into 90 μL of 3% Acetic Acid with Methylene Blue. Mix by pipetting, and count APCs using a hemocytometer and light microscope. Resuspend at 3 × 10⁶ cells/mL.
7. Mix equal volumes of Mitomycin C treated Rag^-/- APCs and purified naïve CD4⁺ T cells. The final ratio of naïve CD4⁺:APCs is 1:3 at 0.5 × 10⁶/mL naïve CD4⁺ T cells, with 1.5 × 10⁶/mL APCs.
8. Add anti-CD3ϵ, anti-CD28, and IL-27 to the CD4⁺:APCs mixture at a final concentration of:
  1. 1 μg/mL anti-CD3ϵ (145-2C11)
  2. 1 μg/mL anti-CD28 (37.51)
  3. 25 ng/mL IL-27
9. Plate 200 mL of cells per well in a 96-well flat bottom plate.
10. Incubate at 37 °C and 5% CO₂.
1. Transduction
  Note: All handling of retroviral production and transduction occurred under a BSL-2 biosafety cabinet.
1. Transduce CD4⁺ T cells after ~18–24 h in culture.
2. Centrifuge the 96-well plate at 1,500 × g and 37 °C for 5 min.
3. Carefully remove 170 μL of media, starting from the top of well to avoid disturbing the cells.
4. Add 20 μL of the concentrated RV (Section A) to the wells.
  Empty parent bicistronic retroviral vectors are used as controls (i.e., pGC-IRES-Thy1.1 for gene of interest-IRES-Thy1.1).
5. Centrifuge the plate at 1,500 × g and 37 °C for 2 h.
6. Place the 96 well plate back in the incubator at 37 °C for 2 h.
7. Add 150 μL of prewarmed media to each well with original culture conditions (described in step C8).
8. Analyze Tr1 cell differentiation, or sort cells for functional assays, 48–72 h post transduction.
1. Analysis of Tr1 Cell Differentiation
1. Prepare flow cytometry staining master mix:
  1. 30 μL of PBS per sample
  2. 1:200 dilution of APC Anti-CD4 Rat [clone: GK1.5], Ghost Dye^TM Violet 510, and anti-reporter antibody (e.g., Pacific Blue Anti-hCD2 [clone:TS1/8], PE anti-Thy1.1 [clone: OX-7])
2. Transfer cells into V-bottom staining plate.
3. Centrifuge cells at 1,000 × g for 1 min.
4. Invert plate to remove supernatant.
5. Add 30 μL of staining master mix to each well, and pipette up and down to resuspend. Incubate at room temperature, protected from light for 20 min.
6. Add 230 μL of PBS to each well. Centrifuge cells at 1,000 × g for 1 min. Invert plate to remove supernatant.
7. Resuspend pellet in 280 μL of PBS. Transfer to flow cytometry tubes for analysis.
8. Use gating strategy in Figure 2, to successfully identify transduced (RV Reporter⁺) and nontransduced (RV Reporter^-) cells. Collect an adequate number of each cell for proper analysis.
  
  Figure 2. Data Collection Gating Strategy. Naïve IL10^GFP/Foxp3^RFP reporter CD4⁺ cells were cultured under Tr1 differentiating conditions and RV transduced with control-Thy1.1. Representative FACS plots of gating strategy for collection of RV transduced Thy1.1⁺ (Reporter⁺) and nontransduced Thy1.1^- (Reporter^-) CD4⁺ cells.
1. Sorting Transduced Tr1 Cells for Functional Assays
1. Combine the cells 48–72 h post transduction in a 15 mL tube.
2. Centrifuge at 600 × g for 5 min. Aspirate supernatant
3. Resuspend in full RPMI media with 1:200 dilution of:
  1. Ghost Dye^TM Violet 510
  2. APC Anti-CD4 Rat [clone: GK1.5]
  3. Anti-Reporter antibody (e.g., Pacific Blue Anti-hCD2 [clone:TS1/8], PE anti-Thy1.1 [OX-7])
  4. Incubate on ice for 15 min, shaking every 5 min.
4. Transfer to 1.5 mL tube. Incubate on ice for 15 min, shaking every 5 min.
5. Add 1 mL of full RPMI media to cells. Centrifuge at 1,500 × g for 1 min, aspirate supernatant.
6. Resuspend cells in 1 mL of full RPMI media, and transfer to tube for sorting.
7. Using the gating strategy from Huang et al. (2017) to sort for Lymphocyte/Singlet/Live/CD4⁺/ RV Reporter⁺/IL10^GFP+Foxp3^RFP-.
  Note: Including Reporter-IL10^GFP+Foxp3^RFP- cells is useful as another control.
8. Proceed to use purified cells in functional assays.
Data analysis
1. Use gating strategy lymphocyte/singlets/live cells/CD4⁺/Reporter⁺CD4⁺ cells for analysis of Tr1 differentiation (Figure 3).
  
  Figure 3. Gating Strategy for Analysis of RV Transduced Tr1 cells. CD4⁺ T cells were transduced with HRas^G12V-IRES-BFP2 RV particles. Representative FACS plots of gating strategy for analysis of RV transduced (BFP⁺) and nontransduced (BFP^-) IL10^GFP+ Foxp3^RFP- Tr1 cells.
2. The frequency of IL10^GFP+Foxp3^RFP-CD4⁺ T cells can be compared via t-test or one-way ANOVA with an appropriate post hoc test in GraphPad Prism, depending on the number of experimental groups. Reporter^- cells can be analyzed as an internal control.
Notes
1. Including the classic Tr1 markers LAG3 and CD49b (Gagliani et al., 2013) provides further evidence of Tr1 differentiation and is highly recommended.
2. Overexpression of transcription factors in the Reporter⁺ cells should be verified via nuclear staining. The eBioscience^TM Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher, 00-5523-00) is recommended.
3. In our hands, the protocol described here produces high efficiency of transduction with several different bicistronic retroviral vector backbone plasmids, such as pGC-IRES-Thy1.1, pMI-IRES-hCD2, and pGC-IRES-BFP2.
4. The efficiency of Plat-E cell transfection (Section A) is a critical step in achieving optimal RV titers and downstream RV transduction efficiency. This can be easily assessed via flow cytometry of Plat-E cells after RV media has been harvested, via gating on viable/Reporter⁺ cells.
5. The size and biological function of an insert affect both Plat-E transfection efficiency and RV transduction efficiency. In our hands, transduction efficiency drops substantially with an insert ~2,500 bp, unless it promotes T cell activation (e.g., constitutively active STAT5).
6. The high efficiency of transduction allows this protocol to be used for dual transduction experiments and transduction of CD8⁺ T cells under IL-10 inducing conditions (Figure 4).
  
  Figure 4. Gating Strategy for Analysis of dual RV Transduced Tr1 cells. Representative FACS plots of IL10^GFP+ Foxp3^RFP- Tr1 cells transduced with HRas^G12V-IRES-BFP2 and caSTAT5-IRES-Thy1.1.
Recipes
1. Full DMEM
  DMEM
  10% FBS
  10 mM HEPES solution (pH 7.5)
  1× Nonessential amino acids
  1 mM Sodium pyruvate
  2 mM L-Glutamine
  1× Penicillin/Streptomycin Solution
2. Full DMEM + Puromycin/Blasticidin
  DMEM
  10% FBS
  10 mM HEPES solution (pH 7.5)
  1× Nonessential amino acids
  1 mM Sodium pyruvate
  2 mM L-Glutamine
  1× Penicillin/Streptomycin Solution
  1 μg/mL Puromycin
  10 μg/mL Blasticidin
3. Full RPMI
  RPMI 1640 Medium
  10% FBS
  10 mM HEPES solution (pH 7.5)
  1× Nonessential amino acids
  1 mM Sodium Pyruvate
  2 mM L-Glutamine
  1× Penicillin/Streptomycin Solution
4. 1× RBC Lysis Buffer
  50 mL of 10× RBC Lysis Buffer
  450 mL of ddH₂O
5. Primary biotin antibodies master mix
  200 μL of full RPMI media per mouse
  Biotin antibodies to a final concentration of:
  5 μg/mL: Anti- B220, CD19, and CD8
  2.5 μg/mL: Anti- Ter119, DX5/CD49b, NK1.1 1, TCRγδ, Gr-1 1, CD122, CD25 CD11c, CD11b, and F4/80
  0.25 μg/mL: Anti-CD44
6. Magnetic anti-biotin microbeads + sorting antibody master mix
  80 μL of full RPMI Media per mouse
  20 μL of anti-biotin microbeads per mouse (Milteny Biotec anti-biotin microbeads)
  1:200 Ghost Dye^TM Violet 510
  1:200 FITC Anti-TCRβ [clone: H57-597]
  1:200 PE Anti-CD25 [clone: PC61.5]
  1:200 APC Anti-CD4 [clone: GK1.5]
  1:200 PE-Cy7 Anti-CD62L [clone: MEL-14]
Acknowledgments
This work is generously supported by National Institutes of Health (R21-AI129422, R56-AI146226, R01-AI138570, and R01-AI151139). M.C.M. is supported by a Careers in Immunology Fellowship from the American Association of Immunologists. W.H. is fellow recipient of the Research Publication Grant in Engineering, Medicine, and Science from the American Association of University Women. We would like to express our gratitude to Drs J. Sun, C. Li, and Y. M. Son for the Plat-E cell line and helpful discussions. We also thank Drs A. Pernis and S. Gupta (pGC) and T. Malek and A. Yu (pMI) for retroviral plasmid backbones. pLKO.3 Thy1.1 (gift from Drs C. Benoist and D. Mathis; Addgene plasmid # 14749) and mTagBFP2-Lifeact-7 (gift from Dr. M. Davidson; Addgene plasmid # 54602) were used to clone Thy1.1 and BFP2 into the respective plasmid backbones, respectively. The protocol presented here was adapted from previously published work (Huang et al., 2017).
Competing interests
M.C.M. declares no conflict of interest. A patent disclosure was filed by W.H. and A.A. on the application of RAS signaling in Tr1 cell differentiation and function at Cornell University, when the original research on RAS signaling was published in 2017. W.H. received funding from MegaRobo Technologies, Ltd and A.A. received funding from 3M, which were not relevant to the research presented in this work.
Ethics
All experimental procedures are approved by the Institutional Biosafety and Animal Care and Use Committees at the Louisiana State University under protocol 18015 (12/10/2018 – present) for biosafety level 2 research, and protocols IACUCAM 18-047 (5/23/2018 – 5/22/2021) and IACUCAM 21-039 (4/26/2021 – 4/25/2024) for animal use.
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