A Simple and Robust Protocol for in vitro Differentiation of Mouse Non-pathogenic T Helper 17 Cells from CD4⁺ T Cells

Materials and Reagents

1. 48-well cell culture plates (ASi Alkali Scientific Inc., catalog number: TP9048)

2. CellPro^TM 70 μm cell strainers (ASi, Falcon^®, catalog number: MT4070)

3. 1 ml syringe (BD Tuberculin Slip Tip, catalog number: 9189520)

4. 50 ml centrifuge tubes (ASi, catalog number: C5602)

5. 15 ml centrifuge tubes (ASi, catalog number: C5600)

6. C57BL/6J mice (The Jackson Laboratory, catalog number: 000664)

7. C57BL/6-IL17a^tm1Bcgen/J mice (The Jackson Laboratory, catalog number: 018472)

8. RBC lysis buffer 10× (Biolegend^®, catalog number: 420302)

9. Cell culture grade water (HyPure^TM, catalog number: SH30529.02)

10. MojoSort^TM Mouse CD4 Naïve T Cell Isolation Kit (Biolegend^®, catalog number: 480040)

11. Coating antibodies:

    1. Anti-mouse CD3ϵ (clone 145-2C11, Bio X Cell, catalog number: BE0001-1)

    2. Anti-mouse CD28 (clone 37.51, Bio X Cell, catalog number: BE0015-1)

12. Staining antibodies:

    1. APC anti-mouse CD4 (clone RM4-5, Biolegend, catalog number: 100516)

    2. PE/Cy7 anti-mouse IL-17A (clone TC11-18H10.1, Biolegend, catalog number: 506922)

13. Cell Stimulation Cocktail Plus Protein Transport Inhibitors (Invitrogen eBioscience^TM, catalog number: 00-4975-93)

14. FoxP3/Transcription Factor Staining Buffer Set (Fixation Diluent, Fixation Reagent, Permeabilization kit, eBioscience^TM, catalog number: 00-5523-00)

15. RPMI 1640 1× with L-glutamine (Corning, catalog number: 10-040-CV)

16. Fetal bovine serum (FBS) (ATLANTA biologicals^TM, R&D SYSTEMS, catalog number: S12450)

17. L-glutamine 200 mM (Corning, catalog number: 25-005-CI)

18. Penicillin-streptomycin solution (PS) 100× (Corning, catalog number: 30-002-CI)

19. Sodium pyruvate 100 mM (Corning, catalog number: 25-000-CI)

20. 2-mercaptoethanol (ME) 1000× (Life Technologies, Gibco^®, catalog number: 21985-023)

21. T_H17 polarization cytokines and antibodies:

    1. Recombinant Human TGF-β1 (PeproTech, catalog number: 100-21C)

    2. Recombinant Murine IL-6 (PeproTech, catalog number: 216-16)

    3. Anti-mouse IFN-γ (clone XMG1.2, Bio X Cell, catalog number: BE0055)

    4. Anti-mouse IL-2 (clone JES6-1A12, Bio X Cell, catalog number: BE0043)

    5. Anti-mouse IL-4 (clone 11B11, Bio X Cell, catalog number: BE0045)

22. Phosphate-buffered saline (PBS) 1× (Corning, catalog number: 21-040-CV)

23. Bovine serum albumin (BSA) (Fisher BioReagents^TM, catalog number: BP1600-100)

24. Ethylenediamine tetraacetate acid (EDTA) 0.5 M (Fisher BioReagents^TM, catalog number: BP2482-500)

25. Carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen^TM, catalog number: C1157)

26. Culture medium (see Recipes)

27. TCR-stimulation antibody mixture (see Recipes)

28. RBC lysis buffer (1×) (see Recipes)

29. MACS buffer (see Recipes)

30. T_H17 polarization medium (see Recipes)

31. Staining buffer (see Recipes)

Equipment

1. NovoCyte (ACEA Biosciences)

Software

1. FlowJo software (BD Biosciences)

Procedure

1. Naïve CD4⁺ T cell isolation and seeding.

   1. Day -2, pre-coat 200 μl TCR-stimulating antibody mixture/well in a 48 well-plate at 4°C.

   2. Day 0, euthanize a C57BL/6J mouse (or IL17A-IRES-GFP-KI mouse), harvest the spleen and lymph nodes (LNs), and isolate naïve CD4⁺ T cells using the MojoSort™ Isolation Kit according to the manufacturer’s instructions.

      Note: Basic information regarding the anatomy of a laboratory mouse can be found at http://www.informatics.jax.org/cookbook/.

   3. Count the cells and seed 2.5 × 10⁵ cells/well in the pre-coated 48-well plate with 500 μl T_H17 polarization medium at 37°C/5% CO₂.

      Note: To follow T cell proliferation, it is optional to stain naive CD4⁺ T cells with CFSE according to the manufacturer’s instructions.

      
      

2. FACS analysis of T_H17 cell differentiation (Figure 1)

   Day 3 (or anytime between day 2 and day 5; we routinely observed over 40% IL17⁺ cells starting at day 2 and remaining for 3 additional days until the media's nutrients are depleted), treat the cells for 4 h with Cell Stimulation Cocktail Plus Protein Transport Inhibitors according to the manufacturer’s for 4 h, and then stain the cells with the anti-mouse CD4 antibody (1:100) for 30 min at 4°C. Next, fix and permeabilize the cells using the FoxP3 Fixation/Permeabilization Kit according to the manufacturer's instructions. Finally, stain the cells with the anti-IL-17A antibody (1:100) at RT for 15 min before FACS analysis (if cultured cells are from an IL17A-IRES-GFP-KI mouse, FACS analysis can be performed directly without the procedures described above in B).

   Note: Basic principles and applications of flow cytometry can be found in the literature (McKinnon, 2018).

   
   

   
   Figure 1. Naïve CD4⁺ T cell isolation and T_H17 differentiation. Flow cytometry data showing that more than 90% naïve CD4⁺ T cells can be obtained after isolation on day 0 (left panel). Day 3, CFSE labeling data showing that more than 50% T_H17 cells can be observed post-polarization (dotted line frame).

Data analysis

FlowJo software: Main gate P1; day 0 data, choose log SSC versus CD4 for gating CD4⁺ T cells. Day 3 data; within P1-CD4 gate (same gating scheme as day 0), choose log CFSE or CD4 versus IL-17A to analyze intracellular IL-17.

Recipes

1. Culture medium

   RPMI 1640 1×

   10% fetal bovine serum (FBS)

   2 mM L-glutamine

   1% sodium pyruvate

   1% PS

   1:1,000 2-ME

   Store at 4°C

2. TCR-stimulation antibody mixture

   10 μg/ml anti-mouse CD3 and 10 μg/ml anti-mouse CD28 antibodies in PBS

3. RBC lysis buffer (1×)

   1 ml 10× buffer with 9 ml cell culture grade water

   Store at 4°C

4. MACS buffer

   500 ml PBS containing 2.5 g bovine serum albumin (BSA) and 2 ml 0.5 M EDTA

   Store at 4°C, fliter using a 0.2-μm filter

5. T_H17 polarization medium

   Culture medium containing mIL-6 (50 ng/ml), hTGF-β1 (20 ng/ml), anti-mIL-2 (8 μg/ml), anti-mIL-4 (8 μg/ml), and anti-mIFN-γ (8 μg/ml)

   Prepare immediately before use

6. Staining buffer

   PBS containing 2% (w/v) BSA

   Store at 4°C

Acknowledgments

The authors would like to acknowledge support by 1R21CA227926-01A1 and 1UO1CA232488-01 from the National Institutes of Health (Cancer Moonshot program), 1R01AI114581 from the National Institutes of Health, V2014-001 from the V-Foundation, and 128436-RSG-15-180-01-LIB from the American Cancer Society (to RW). With respect, the authors wish to emphasize that this protocol builds upon previous knowledge and experimental details of T_H17 in vitro differentiation that has been established in the field for many years (Harrington, Hatton et al., 2005; Park, Li et al., 2005; Veldhoen; Hirota et al., 2009). This protocol was adapted with minor modification from previous study published by Wu et al. (2020).

Competing interests

The authors declare no conflicts of interest.

References

1. Awasthi, A., Riol-Blanco, L., Jager, A., Korn, T., Pot, C., Galileos, G., Bettelli, E., Kuchroo, V. K. and Oukka, M. (2009). Cutting edge: IL-23 receptor gfp reporter mice reveal distinct populations of IL-17-producing cells. J Immunol 182(10): 5904-5908.
2. Bettelli, E., Korn, T., Oukka, M. and Kuchroo, V. K. (2008). Induction and effector functions of T_H17 cells. Nature 453(7198): 1051-1057.
3. Bhaumik, S. and Basu, R. (2017). Cellular and Molecular Dynamics of Th17 Differentiation and its Developmental Plasticity in the Intestinal Immune Response. Front Immunol 8: 254.
4. Cua, D. J., Sherlock, J., Chen, Y., Murphy, C. A., Joyce, B., Seymour, B., Lucian, L., To, W., Kwan, S., Churakova, T., Zurawski, S., Wiekowski, M., Lira, S. A., Gorman, D., Kastelein, R. A. and Sedgwick, J. D. (2003). Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421(6924): 744-748.
5. Gaffen, S. L., Hernandez-Santos, N. and Peterson, A. C. (2011). IL-17 signaling in host defense against Candida albicans. Immunol Res 50(2-3): 181-187.
6. Guglani, L. and Khader, S. A. (2010). Th17 cytokines in mucosal immunity and inflammation. Curr Opin HIV AIDS 5(2): 120-127.
7. Hartigan-O'Connor, D. J., Hirao, L. A., McCune, J. M. and Dandekar, S. (2011). Th17 cells and regulatory T cells in elite control over HIV and SIV. Curr Opin HIV AIDS 6(3): 221-227.
8. Jager, A., Dardalhon, V., Sobel, R. A., Bettelli, E. and Kuchroo, V. K. (2009). Th1, Th17, and Th9 effector cells induce experimental autoimmune encephalomyelitis with different pathological phenotypes. J Immunol 183(11): 7169-7177.
9. Korn, T., Bettelli, E., Oukka, M. and Kuchroo, V. K. (2009). IL-17 and Th17 Cells. Annu Rev Immunol 27: 485-517.
10. Lee, Y., Awasthi, A., Yosef, N., Quintana, F. J., Xiao, S., Peters, A., Wu, C., Kleinewietfeld, M., Kunder, S., Hafler, D. A., Sobel, R. A., Regev, A. and Kuchroo, V. K. (2012). Induction and molecular signature of pathogenic TH17 cells. Nat Immunol 13(10): 991-999.
11. Lee, Y., Collins, M. and Kuchroo, V. K. (2014). Unexpected targets and triggers of autoimmunity. J Clin Immunol 34 Suppl 1: S56-60.
12. Ouyang, W., Kolls, J. K. and Zheng, Y. (2008). The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity 28(4): 454-467.
13. Katherine M McKinnon (2018). Flow Cytometry: An Overview. Curr Protoc Immunol. 120:5.1.1-5.1.11.
14. Harrington, L. E., R. D. Hatton, P. R. Mangan, H. Turner, T. L. Murphy, K. M. Murphy and C. T. Weaver (2005). Interleukin 17-producing CD4⁺ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol 6(11): 1123-1132.
15. Park, H., Z. Li, X. O. Yang, S. H. Chang, R. Nurieva, Y. H. Wang, Y. Wang, L. Hood, Z. Zhu, Q. Tian and C. Dong (2005). A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol 6(11): 1133-1141.
16. Veldhoen, M., K. Hirota, J. Christensen, A. O'Garra and B. Stockinger (2009). Natural agonists for aryl hydrocarbon receptor in culture medium are essential for optimal differentiation of Th17 T cells. J Exp Med 206(1): 43-49.
17. Ruohan Wu*, Xuyong Chen*, Siwen Kang*, Tingting Wang, JN Rashida Gnanaprakasam, Yufeng Yao, Lingling Liu, Gaofeng Fan, Mark R. Burns, Ruoning Wang†. (2020). De novo synthesis and salvage pathway coordinately regulate polyamine homeostasis and determine T cell proliferation and function. Science Advances 6 (51): eabc4275.