Th9

Th9 Polarization of Mouse CD4⁺ Cells

Introduction:

Th9 cells are a subpopulation of T helper cells involved in allergic diseases and resistance against intestinal nematodes. Unlike Treg cells, Th9 cells are not suppressive, as they can promote T cell proliferation along with other effector T cells. Th9 cells do not express any well-defined transcription factors like T-bet, GATA3, RORγt, or FOXP3, clearly differentiating them from Th1, Th2, Th17 and FOXP3+ iTreg populations. TGF-β reprograms Th2 T helper cells to lose their characteristic profile of IL-4, IL-5 and IL-13 secretion and switch to IL-9 secretion. Differentiation of Th9 cells can be obtained by a combination of TGF-β and IL-4. In addition, the neutralization of IFN-γ is critical to drive the pathway to Th9 cells. Here we provide an effective protocol for the generation of mouse Th9 cells in vitro.

Isolation of CD4+ Cells From Lymph Nodes:

1. Harvest lymph nodes (superficial cervical, mandibular, axillary, inguinal, and mesenteric) from mice.
2. Tease lymph nodes through a sterile 70-µm nylon cell strainer to obtain single-cell suspensions in IMDM containing 10% FCS (complete medium).
3. Resuspend cells in complete medium and use your favorite method to isolate CD4⁺ cells, such as the MojoSort™ Mouse CD4 T Cell Isolation Kit.

Th9 Polarization of CD4⁺ Cells:

1. On day 0, coat 60 × 15 mm of plastic petri dishes with anti-mouse CD3ε, clone 145-2C11 (5 µg/ml). Incubate at 37°C for 2 hours or 4°C overnight. Aseptically decant antibody solution from the plate. Wash plate 3 times with sterile PBS. Discard liquid.
2. Plate CD4⁺ cells at 10 x10⁶/5 ml/dish. Culture cells for 3 days in the presence of anti-mouse CD28, clone 37.51 (5 µg/mL), recombinant human TGF-β1 (10 ng/mL), recombinant mouse IL-4 (10 ng/mL), recombinant mouse IL-2 (20 ng/ml), and anti-mouse IFN-γ, clone XMG1.2 (10 µg/mL).
3. On day 3, wash cells once and then restimulate in complete medium with 500 ng/ml PMA and 500 ng/mL ionomycin, in the presence of monensin for 6 hours.
4. After harvesting, the cells are ready for staining.

*Note: recombinant human TGF-β is effective for stimulating mouse cells.

Reagent List:

• Sterile PBS
• Cell culture medium (IMDM supplemented with 10% FBS)
• Sterile plastic petri dishes
• RBC Lysis Buffer (Cat. No. 420301)
• Anti-mouse CD3ε Antibody, clone 145-2C11 (Ultra-LEAF™ format, Cat. No. 100340)
• Anti-mouse CD28, clone 37.51 (Ultra-LEAF™ format, Cat. No. 102116)
• Anti-mouse IFN-γ, clone XMG1.2 (Ultra-LEAF™ format, Cat. No. 505834)
• Recombinant mouse IL-2 (carrier-free) (Cat. No. 575402)
• Recombinant mouse IL-4 (carrier-free) (Cat. No. 574302)
• Recombinant human TGF-β1 (carrier-free) (Cat. No. 781802)
• Monensin Solution (Cat. No. 420701)
• PMA (Phorbol 12-myristate 13-acetate) (Cat. No. P8139 from Sigma)
• Ionomycin (Cat. No. I0634 from Sigma)

References:

1. Soler, D. et al. 2006. J Immunol. 117:6940.
2. Schmitt, E. et al. 1994. J Immunol. 153:3989.
3. Darhalhon, V. et al. 2008. Nat. Immunol. 9:1347.

Data:

Naïve BALB/c mouse CD4⁺ T cells were polarized with 5 µg/ml plate-bound anti-mouse CD3ε (clone 145-2C11 , Cat. No. 100339), 5 µg/ml soluble anti-mouse CD28 (clone 37.51, Cat. No. 102115), 20 ng/ml IL-2 (Cat. No. 575402), 10 ng/ml IL-4 (Cat. No. 574302), 10 ng/ml TGF-β1 (Cat. No. 781802), and 10 µg/ml anti-mouse IFN-γ (clone XMG1.2, Cat. No. 505833) for 3 days. On day 3, the cells were re-stimulated with 500 ng/ml PMA/ionomycin in the presence of monensin for 6 hours. Then the cells were harvested and surface stained with CD4 PE (clone GK1.5, Cat. No. 100408), intracellular stained with IL-9 APC (clone RM9A4, Cat. No. 514105) along with IFN-γ Alexa Fluor® 488 (clone XMG1.2, Cat. No. 505813), IL-4 Alexa Fluor® 488 (clone 11B11, Cat. No. 504109), or IL-17 Alexa Fluor® 488 (clone TC11-18H10.1, Cat. No. 506910) after fixation and permeabilization.