Th1

Th1 Polarization of Mouse CD4⁺ Cells

Introduction:

Th1 cells are a distinct lineage of CD4⁺ effector T cells that are characterized by their production of IL-2, IFN-γ and Lymphotoxin (LT) after activation in the presence of IL-12. IL-12 drives CD4⁺ T cell differentiation to the Th1 lineage through the sequential involvement of cell-extrinsic and cell-intrinsic factors, including signal transducer and transcription activator 1 (STAT1), the transcription factor T-bet, IL-12 and STAT4. Th1 cells are characterized by the expression of the cell surface markers CCR5, CXCR3, cytokine receptors IL-12Rβ2, and IL-18Rα. They are mainly involved in the cellular immune response and host defense against intracellular pathogens. They are also found to be associated with autoimmune diseases, including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, type 1 diabetes, organ-specific autoimmune disorders, Crohn's disease, Helicobacter pylori-induced peptic ulcer, acute kidney allograft rejection, and unexplained recurrent abortions.

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.

Th1 Polarization of CD4⁺ Cells:

1. On day 0, coat 12-well plate with anti-mouse CD3ε, clone 145-2C11 (3 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 1.0 x 10⁶/1ml/well. Culture cells for 5 days at 37°C, 5% CO2, in the presence of anti-mouse CD28, clone 37.51 (3 µg/mL), anti-mouse IL-4, clone 11B11 (10 µg/mL), recombinant mouse IL-2 (5 ng/mL), and recombinant mouse IL-12 (10 ng/ml).
3. On day 3, if media is yellow, add 2 ml/well of fresh media.
4. On day 5, wash cells once and then restimulate in complete media with 50 ng/ml PMA, 1 µg/ml ionomycin and 10 µl monensin (1000x), in a 6-well plate in incubator at 37°C for 5 hours.
5. After harvesting, the cells are ready for staining.

Reagent List:

• Sterile PBS
• Cell culture medium (RPMI 1640 supplemented with 10% FBS)
• Sterile 12-well plate
• Sterile 6-well plate
• 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 IL-4, clone 11B11 (Ultra-LEAF™ format, Cat. No. 504122)
• Recombinant mouse IL-2 (carrier-free) (Cat. No. 575402)
• Recombinant mouse IL-12 (p70) (carrier-free) (Cat. No. 577002)
• Monensin Solution (Cat. No. 420701)
• PMA (Phorbol 12-myristate 13-acetate) (Cat. No. P8139 from Sigma)
• Ionomycin (Cat. No. I0634 from Sigma)

References:

1. Bo Jin. et al. 2012. Clin Dev Immunol. 2012: 836485
2. Zhu J. et al. 2008. Blood. 112: 1557
3. Nakae S. et al. 2007. JLB. 1:1189
4. Romagnani S. 2000. Ann Allergy Asthma Immunol. 85: 9
5. Rosloniec E. 2002. Arthritis Res. 2002. 4:333

Data:

Naïve BALB/c mice CD4⁺ T cells were polarized with 3 µg/ml plate-bound anti-mouse CD3 (clone 145-2C11, Cat. No. 100339), 3 µg/ml soluble anti-mouse CD28 (clone 37.51, Cat. No. 102115), 10 ng/ml IL-12 (Cat. No. 577002), 5 ng/ml IL-2 (Cat. No. 575402), and 10 µg/ml anti-mouse IL-4 (clone 11B11, Cat. No. 504121) for 5 days and fresh media was added on day 3. On day 5, the cells were re-stimulated with 50 ng/ml PMA, 1 µg/ml ionomycin in the presence of monensin for 5 hours. Then the cells were harvested and surface stained with CD4 PerCP-Cy5.5 (clone RM4-5, Cat. No. 100539), intracellular stained with IFN-γ FITC (clone XMG1.2, Cat. No. 505805), IL-4 APC (clone 11B11, Cat. No. 504105) and IL-17 PE (clone TC11-18H10.1, Cat. No. 506903) after fixation and permeabilization.