Materials and Reagents

Pipette tips
0.22 μm 1,000 ml Stericup (Millipore Sigma, catalog number: SCGPU11RE)
0.22 μm 50 ml Steriflip (Millipore Sigma, catalog number: SCGP00525)
LS Columns (Miltenyi Biotec, catalog number: 130-042-101)
50 ml Falcon conical tube (Corning, catalog number: 352070)
15 ml CentriStar conical tube (Corning, catalog number: 430790)
5 ml Falcon Round Bottom test tube (Corning, catalog number: 352052)
96-well round bottom, non-TC treated plates (Sarstedt, catalog number: 82.1582.001)
CytoOne Non-treated 6-well plates (USA Scientific, catalog number: CC7672-7506)
Falcon 70 μm cell stainer (Corning, catalog number: 352350)
Plain and frosted glass slides (VWR International, catalog number: 48312-004)
Foxp3^GFP(B6.Cg-Foxp3tm2(EGFP)Tch/J) mice (THE JACKSON LABORATORY, catalog number: 006772)
The best T_regP cell yields come from younger (5-6 weeks old) female mice due to a generally large thymus. If younger mice are not available older mice will provide usable T_regP cells. However, the number of T_regP cells recovered could be reduced by 50% of that obtained from younger mice.
Recombinant Human IL-2 (R&D Systems, catalog number: 202-IL)
Anti-mouse CD4 (GK1.5) BV786 (1:200) (BD Biosciences, catalog number: 563331)
Anti-mouse CD8a (53-6.7) Biotin (1:200) (Tonbo Biosciences, catalog number: 30-0081-U500)
Anti-mouse CD25 (PC61.5) PerCP-Cy5.5 (1:200) (Tonbo Biosciences, catalog number: 65-0251-U100)
Anti-mouse CD73 (eBioTY/11.8) eF450 (1:100) (eBioscience, catalog number: 48-0731-82)
Anti-mouse Ter119 (Ter-119) Biotin (1:200) (BD Biosciences, catalog number: 553672)
(Optional) antibodies:
1. Anti-CD11b (M1/70) Biotin (1:200) (Tonbo Biosciences, catalog number: 30-0112-U500)
2. Anti-CD11c (N418) Biotin (1:200) (Tonbo Biosciences, catalog number: 30-0114-U500)
3. Anti-NK1.1 (PK136) Biotin (1:200) (Tonbo Biosciences, catalog number: 30-5941-U500)
4. Anti-γδTCR (eBioGL3) Biotin (1:200) (eBioscience, catalog number: 13-5711-85)
5. Anti-B220 (RA3-6B2) Biotin (1:200) (Tonbo Biosciences, catalog number: 30-0452-U500)
Streptavidin APC-eF780 (1:200) (eBioscience, catalog number: 47-4317-82)
Viability Dye Ghost^TM Red 780 (1:1,000) (Tonbo Biosciences, catalog number: 13-0865-T100)
Streptavidin Microbeads (Miltenyi Biotec, catalog number: 130-048-101)
AccuCheck Counting Beads (Life Technologies, catalog number: PCB100)
Sodium azide (NaN₃) (Ricca Chemical, catalog number: 7144.8-16)
10x PBS (Fisher BioReagents, catalog number: BP399-20)
Ethylenediaminetetraacetic Acid (EDTA) (Fisher Scientific, catalog number: BP120-500)
Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S11150)
RPMI 1640 media (Corning, catalog number: 15-040-CV)
Penicillin-Streptomycin Solution (Corning, catalog number: 30-002-CL)
L-Glutamine (Corning, catalog number: 25-005-CL)
MEM Nonessential amino acids (Corning, catalog number: 25-025-CL)
2-mercaptoethanol (MP Bio, catalog number: 02194705-CF)
HEPES (Corning, catalog number: 25-060-CL)
Sodium Pyruvate (Corning, catalog number: 25-000-CL)
Sort Buffer (see Recipes)
FACS Buffer (see Recipes)
cRPMI (see Recipes)

Equipment

Pipettes
Sorvall Legend X1R Centrifuge (Thermo Scientific, catalog number: 75004260)
BD Biosciences LSR II (BD, model: LSR II)
BD Biosciences FACSAria II (BD, model: FACSAria II)
MACS Multistand (Miltenyi Biotec, catalog number: 130-042-303)
QuadroMACS Separator Magnet (Miltenyi, catalog number: 130-090-976)
Class II Biosafety Cabinet/Laminar Flow Hood
IncuSafe Incubator (Panasonic, catalog number: KM-CC17T0A)
Hemocytometer (Fisher, catalog number: 0267110)
Refrigerator (2-8 °C, Kenmore)
Panasonic -80 °C VIP Plus Freezer

Software

FlowJo 10 (10.5.3) (https://www.flowjo.com/)

Procedure

T_regP cell isolation
1. Euthanize mice by CO₂ inhalation.
2. Harvest thymi from mice into 5 ml Sort buffer in 6-well plates. The number of conditions tested and replicates determine the number of mice required for each experiment. In a simple experiment, 1 mouse can be used as this should provide 5-10 x 10⁴ CD25⁺ and Foxp3^lo T_regP cells.
3. Work in a clean biosafety cabinet from this point on.
4. Mechanically dissociate thymi between frosted glass slides or favored method of mechanical dissociation.
5. Transfer cells into 50 ml conical vial through 70 μm filter.
6. Spin cells at 350 x g for 10 min, at 4 °C (keep thymi in individual 50 ml conical tubes).
7. Resuspend cells in 1 ml depletion cocktail of biotin-labeled antibodies. This cocktail of antibodies should include anti-CD8 (1:200) and anti-Ter119 (1:200). However other optional depletion antibodies can be added to this cocktail such as anti-CD11b (1:200), anti-CD11c (1:200), anti-NK1.1 (1:200), anti-γδTCR (1:200) and anti-B220 (1:200).
8. Incubate on ice for 20 min and cover to protect from light.
9. Add 15 ml Sort buffer; spin cells at 350 x g for 5 min, at 4 °C.
10. Count the cells and resuspend in Strepavidin microbead cocktail (~1 μl beads per 2-3 million cells diluted in Sort buffer to a final volume of ~400 μl/thymus).
11. Incubate for 20 min at 4 °C (agitate by swirling tube gently after 10 min).
12. Add 10 ml Sort buffer; spin cells at 350 x g for 5 min, at 4 °C.
13. Pre-rinse LS columns (1 column/thymus) with 3 ml cold Sort Buffer.
14. Resuspend cells in 2 ml Sort buffer.
15. Apply cells from individual thymus to separate LS columns on QuadroMACS Miltenyi separator magnet through a filter (Figure 1).
  
  Figure 1. Schematic of in vitro T_regP differentiation assay workflow. First, thymi are processed and enriched for CD4 single positive (CD4SP) thymocytes. This fraction is stained for surface markers then each T_regP is isolated by FACS. Isolated T_regP are stimulated with cytokine in 96-well plate for a pre-determined time. These cells are then harvested and analyzed by flow cytometry for upregulation of Foxp3 and/or CD25.
16. Wash columns 3 times each with 3 ml cold Sort buffer.
17. Collect unbound fraction in 15 ml conical vials; spin cells at 350 x g for 5 min, at 4 °C.
18. Combine cells into a single 15 ml conical tube; apply surface staining cocktail [CD4 (1:200), Live-Dead (1:1,000), CD25 (1:200), CD73 (1:100) and Streptavidin-APC-eF780 (1:200)]. The Streptavidin-APC-eF780 is used to detect biotin-conjugated antibodies used for magnetic depletion, including CD8, Ter119 and any other optional antibodies included from Step 7 above.
19. Incubate on ice for 20 min and cover to protect from light.
20. Add 14 ml Sort buffer; spin cells at 350 x g for 5 min, at 4 °C.
21. Resuspend cells in 1 ml Sort buffer and filter cells through a 70 μm filter into a new, pre-chilled 15 ml conical; wash the filter with Sort buffer to bring cells to appropriate final volume.
22. Set up gates on FACS Aria II for sorting T_regP cells as described in Figure 2.
23. Collect CD25⁺ and/or Foxp3^lo T_regP cells into separate 5 ml round bottom tubes with 1.5 ml Catch buffer (1x PBS + 50% FBS).
  
  Figure 2. Flow cytometry gating strategy for sorting T_regP. A. Flow cytometry gating scheme for sorting T_regP from CD8/Ter119 depleted thymocytes. DUMP channel contains CD8, Ter119 (these are labeled with biotin conjugated antibodies during magnetic depletion that are probed with APC-eF780-conjugated streptavidin) and Live-Dead. CD73⁺ cells represent recirculating and long-term resident T_regP and T_reg. Thus sorting CD73^- thymocytes ensures analysis of de novo developing T_regP. B. Post-sort purity analysis of CD25⁺ TregP (top) and Foxp3^lo T_regP (bottom).
Stimulation of T_regP cells
1. Make stimulation media (example: 2 U/ml IL-2 in cRPMI for a final concentration of 1 U/ml).
2. Plate 100 μl of stimulation media into each well of non-TC treated round bottom 96-well plate.
3. Place the 96-well plate in 37 °C incubator.
4. Fill 5 ml round bottom tubes containing sorted T_regP cells with cRPMI, spin cells at 350 x g for 5 min, at 4 °C.
5. Resuspend cells at 1-2 x 10⁵ cells/ml in cRPMI.
6. Add 100 μl of cells to 100 μl stimulation media in 96-well plates (plate 1-2 x 10⁴ cells/well).
7. Place the 96-well plates into 37 °C incubator.
8. Incubate for 24-72 h (we perform these assays for 72 h typically but CD25 and/or Foxp3 upregulation can be observed at earlier timepoints).
Analysis of in vitro T_regP differentiation assay
1. Add 100 μl FACS buffer to each well of stimulated T_regP.
2. Spin cells at 350 x g for 5 min at 4 °C.
3. Decant supernatant and add 200 μl FACS buffer.
4. Spin cells at 350 x g for 5 min at 4 °C.
5. Resuspend cells in 50 μl staining cocktail [anti-CD4 (1:200), anti-CD8 (1:200), anti-CD25 (1:200), Live-Dead (1:1,000)].
6. Incubate on ice for 20 mins and cover to protect from light.
7. Add 200 μl FACS buffer.
8. Spin cells at 350 x g for 5 min at 4 °C.
9. Resuspend each sample in FACS buffer/counting bead master mix (190 μl FACS buffer + 10 μl counting beads/sample).
10. Analyze differentiation of T_regP to mature T_reg cells (typically co-expression of CD25 and Foxp3).

Data analysis

Data analysis was performed in FlowJo 10.
Gating scheme for quantification of T_reg differentiation is shown in Figure 3. Briefly, lymphocytes were gated followed by singlets. CD4⁺, CD8/Live-Dead^- singlets were gated and displayed for expression of CD25 and Foxp3. Representative data can be found in Owen et al. (2019) in Figure 7A, Supplemental Figure 1 and Supplemental Figure 7.

Figure 3. Flow cytometry gating scheme for analysis of T_regP following 3 days of stimulation. Media alone (left) fails to convert T_regP to T_reg cells while 1 U/ml IL-2 stimulation (right) converts T_regP to T_reg cells. Beads are used to calculate total numbers of T_reg cells generated in each sample. The formula used is Total cells = Cells detected x (Known Bead number added/Beads detected).
Published results (Burchill et al., 2008; Lio and Hsieh, 2008; Vang et al., 2008; Mahmud et al., 2014; Owen et al., 2019) have indicated successful conversion of CD25⁺ or Foxp3^lo T_regP is represented by dual expression of CD25 and Foxp3 in response to IL-2. However, other cytokines we have tested, such as IL-4 (Owen et al., 2019), have led to generation of CD25⁺ Foxp3⁺ T_reg cells and the maintenance of CD25^-Foxp3^lo cells. Interestingly, IL4 stimulation of CD25^-Foxp3^lo T_regP cells results in the development of CD25⁺Foxp3⁺ T_reg cells that express distinctly lower levels of CD25 than observed with IL-2 stimulation. Thus, depending on the condition, it is relevant to also consider the proportion of CD25^-Foxp3⁺ cells as well as the mean fluorescence intensity of CD25 and Foxp3, as shown in Owen et al. (2019) Supplemental Figure 7.

Notes

In our hands, T_regP cells are a delicate population and quite susceptible to cell death. Given this, gentle processing throughout the cell isolation protocol will yield higher amounts of T_regP cells from thymi.
A typical yield from 1 thymus is ~50,000-100,000 T_regP depending on age (5-6 weeks being ideal) and sex (female having larger thymi).
We make fresh cRPMI for each assay to help maintain viability of T_regP cells in vitro.
Our greatest success has been using round bottom, non-TC treated 96-well plates. Other 96-well formats led to increased cell death in our hands.

Recipes

Sort Buffer (store at 4 °C, ~55-60 ml/sample)
1x PBS
2% FBS
2 mM EDTA
FACS Buffer (store at 4 °C, ~1 ml/well)
1x PBS
2% FBS
2 mM EDTA
0.05% NaN₃
cRPMI (filter sterilize, store at 4 °C)
RPMI 1640 Medium
10% Fetal Bovine Serum
1% Penicillin/Streptomycin
1% L-Glutamine
1% Sodium Pyruvate
1% Non-essential amino acids
10 mM HEPES
50 μM 2-mercaptoethanol

Acknowledgments

This work was supported by R01AI124512 (MAF) and an Immunology training grant 2T32AI007313 (DLO) from the NIH. This protocol is adapted from methods originally published by Lio and Hsieh (2008) and modified for studies from our lab (Burchill et al., 2008; Vang et al., 2008; Mahmud et al., 2014; Owen et al., 2019).

Competing interests

The authors declare no competing financial or non-financial interests.

Ethics

All animal experimentation in this protocol was approved by the University of Minnesota Institutional Animal Care and Use Committee (most recent IACUC protocol 1904-36975A, valid 5/16/2019-5/15/2022).

References

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Lio, C. W. and Hsieh, C. S. (2008). A two-step process for thymic regulatory T cell development. Immunity 28(1): 100-111.
Mahmud, S. A., Manlove, L. S., Schmitz, H. M., Xing, Y., Wang, Y., Owen, D. L., Schenkel, J. M., Boomer, J. S., Green, J. M., Yagita, H., Chi, H., Hogquist, K. A. and Farrar, M. A. (2014). Costimulation via the tumor-necrosis factor receptor superfamily couples TCR signal strength to the thymic differentiation of regulatory T cells. Nat Immunol 15(5): 473-481.
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Vang, K. B., Yang, J., Mahmud, S. A., Burchill, M. A., Vegoe, A. L. and Farrar, M. A. (2008). IL-2, -7, and -15, but not thymic stromal lymphopoeitin, redundantly govern CD4⁺Foxp3⁺ regulatory T cell development. J Immunol 181(5): 3285-3290.