Th2

Th2 Polarization of Mouse CD4⁺ T Cells

Introduction:

Th2 cells are a distinct lineage of CD4⁺ effector T cells that secrete IL-4, IL-5, IL-9 and IL-13 after activation in the presence of IL-4 and either IL-2, IL-7, or Thymic Stromal Lymphopoietin (TSLP). IL-4 drives CD4⁺ T cells by engaging the IL-4R, thereby activating STAT6 and the transcription factor GATA-3. Th2 cells are characterized by the expression of the cell surface receptors, ST2/IL-1 R4, CXCR4, CCR3, CCR4, and CCR8. These cells are responsible for strong antibody production, eosinophil activation, and inhibition of several macrophage functions. They mediate development and maintenance of many allergic diseases such as allergic airway inflammation (allergic asthma) or atopic dermatitis, Omenn's syndrome, reduced protection against some intracellular pathogens, transplantation tolerance, chronic graft vs host disease, atopic disorders, and some systemic autoimmune diseases.

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 complete RPMI 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.

Th2 Polarization of CD4⁺ Cells:

1. On day 0, plate CD4⁺ cells at 30 x10⁶/30 ml/T-75 flask. Culture cells for 3 days in complete RPMI containing 10% FCS, Con A (5 µg/mL), recombinant mouse IL-2 (20 ng/ml), and recombinant mouse IL-4 (50 ng/ml).
2. On day 3, harvest the cells and wash cells once. Seed 15 x10⁶ cells/30 ml/T-75 flask along with recombinant mouse IL-2 (20 ng/ml) and recombinant mouse IL-4 (50 ng/ml).
3. On day 5, coat a 60 x 15 mm tissue culture petri dish with anti-mouse CD3ε, clone 145-2C11, 10 µg/mL in PBS, 5 ml/dish. Incubate in a 4°C refrigerator overnight.
4. On day 6, wash the anti-mouse CD3ε pre-coated tissue culture petri dish with PBS. Harvest the cells from the flask (that were seeded on Day 5), wash them twice, and seed at 20 x10⁶/10 ml/petri dish along with 10 µl of monensin (1000x) and anti-mouse CD28, clone 37.51 (5 µg/ml). Incubate for 6 hours at 37°C in a CO₂ incubator.
5. After harvesting, the cells are ready for staining.

Reagent List:

• Sterile PBS
• Cell culture medium (RPMI 1640 supplemented with 10% FBS)
• Sterile plastic petri dishes
• Sterile T-75 culture flask
• RBC Lysis Buffer (Cat. No. 420301)
• Concanavalin A (Con A) (Sigma, Cat. No. C5275)
• 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)
• Recombinant mouse IL-2 (carrier-free) (Cat. No. 575402)
• Recombinant mouse IL-4 (carrier-free) (Cat. No. 574302)
• Monensin Solution (Cat. No. 420701)

References:

1. Romagnani S. 2000. Ann Allergy Asthma Immunol. 85: 9
2. Tsuchiya T. 2004. Blood. 103:236
3. Paul WE. 2010. Immunol Cell Biol. 88: 236
4. William E. et al. 2010. Nat Rev Immunol. 10: 225
5. Paul WE. 2010. Nat Rev Immunol. 4: 225

Data:

Naïve BALB/c mice CD4⁺ T cells were polarized with 5 µg/ml ConA, 20 ng/ml mouse IL-2 (Cat. No. 575402), and 50 ng/ml mouse IL-4 (Cat. No. 574302) for 3 days. The cells were cultured for an additional 3 days with fresh media and IL-2/IL-4. On day 6, the cells were re-stimulated with plate-bound 10 µg/ml anti mouse CD3ε (clone 145-2C11, Cat. No. 100339), soluble 5 µg/ml anti-mouse CD28 (clone 37.51, Cat. No. 102115) in the presence of monensin for 6 hours. Then the cells were harvested and surface stained with CD4 PerCP/Cy5.5 (clone RM4-5, Cat. No. 100539), intracellular stained with IL-4 APC (clone 11B11, Cat. No. 504105) along with FOXP3 Alexa Fluor® 488 (clone MF14, Cat. No. 126405), IFN-γ Alexa Fluor® 488 (clone XMG1.2, Cat. No. 505815), or IL-17A Alexa Fluor® 488 (clone TC11-18H10.1, Cat. No. 506909) after fixation and permeabilization.