Activation Bundles

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.

Treg Polarization of Mouse CD4⁺ Cells

Introduction:

Regulatory T cells (Tregs) are a subpopulation of T cells dedicated to maintaining self-tolerance that express the forkhead family transcription factor FOXP3 (forkhead box p3). FOXP3⁺ Tregs mainly comprise naturally occurring Tregs (nTregs) and adaptive or induced Tregs (aTreg or iTreg). nTreg cells arise in the thymus following ligation of high-affinity T cell receptors (TCRs). These cells express the high-affinity form of the interleukin-2 (IL-2) receptor but depend on exogenous IL-2 to maintain FOXP3 expression. iTreg cells differentiate from mature, naïve CD4⁺ T cells in peripheral lymphoid organs and other tissue upon cellular activation in the presence of TGF-β1. In vitro, a combination of IL-2 and anti-CD3/CD28 antibodies sufficiently expand Tregs. Expansion of Tregs can be further enhanced by artificial antigen presenting cells over-expressing CD86 and FcR. Treg cells play an indispensable role in maintaining immunological unresponsiveness to self-antigens and in suppressing excessive immune responses deleterious to the host. Tregs exert their function by secretion of immunosuppressive soluble factors such as IL-9, IL-10 and TGF-β and cell-mediated regulation via the high affinity TCR, cytolytic activity, and other co-stimulatory molecules such as GITR and CTLA-4.

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.

Treg 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), recombinant mouse IL-2 (5 ng/mL), and recombinant human TGF-β1 (5 ng/ml).
3. On day 3, if media is yellow, add 2 ml/well of fresh media.
4. On day 5, 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 (RPMI 1640 supplemented with 10% FBS)
• Sterile 12-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)
• Recombinant mouse IL-2 (carrier-free) (Cat. No. 575402)
• Recombinant human TGF-β1 (carrier-free) (Cat. No. 781802)
• Monensin Solution (Cat. No. 420701)

References:

1. Chai JG. et al. 2008. J Immunol. 180: 858.
2. Papatriantafyllou M. et al. 2011. Nat. Rev. Immunol. 11:500.
3. Thomas D. 2012. Blood. 19: 4430.
4. Bluestone J. 2003. Nat. Rev. Immunol. 3: 253.
5. Eisenstein E. et al. 2009. Infection and Immunity. 65: 26R.

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 CD28.2, Cat. No. 102115), 5 ng/ml IL-2 (Cat. No. 575402), and 5 ng/ml TGF-β1 (Cat. No. 781802) for 5 days and fresh media was added on day 3. Cells were then harvested and surface stained with CD4 PerCP/Cy5.5 (clone RM4-5, Cat. No. 100539), intracellular stained with FOXP3 Alexa Fluor® 647 (clone 150D, Cat. No. 320013), IFN-γ FITC (clone XMG1.2, Cat. No. 505805) or IL-4 APC (clone 11B11, Cat. No. 504105) after fixation and permeabilization.

Th17 Polarization of Mouse CD4⁺ Cells

Introduction:

T helper 17 (Th17) cells are a subset of CD4⁺ T helper cells characterized by their production of IL-17, particularly IL-17A and IL-17F. They are involved in autoimmune diseases such as multiple sclerosis, psoriasis, autoimmune uveitis, rheumatoid arthritis, and juvenile diabetes, but are also important in anti-microbial defenses at epithelial/mucosal barriers, particularly against Candida and Staphylococcus. In addition to IL-17, Th17 cells also produce IL-21 and IL-22 that contribute to pro-inflammatory responses. The transcription factors RORα and RORγt have been identified as important factors in the differentiation of these cells. Th17 cells can be induced in vitro by a variety of cytokines including TGF-β, IL-6, IL-21 and IL-23. Here we provide an effective protocol for the generation of mouse IL-17 producing 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 complete RPMI containing 10% FCS and 0.05 mM 2-ME (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.

Th17 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 x 10⁶/5 ml/dish. Culture cells for 4 days in the presence of anti-mouse CD28, clone 37.51 (5 µg/mL), recombinant mouse IL-6 (50 ng/mL), recombinant human TGF-β1 (1 ng/mL), recombinant mouse IL-23 (5 ng/ml), anti-mouse IL-4 (10 µg/mL), and anti-mouse IFN-γ (10 µg/mL).
3. On day 3, slowly add 5 ml of fresh media along with same the concentration of antibodies/cytokines as used on day 0.
4. On day 4, wash cells once and then restimulate in complete medium with 500 ng/ml PMA and 500 ng/mL ionomycin, in the presence of Brefeldin A (If you are looking for IL-21 production, use monensin) for 4 - 5 hours.
5. 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 (RPMI 1640 supplemented with 10% FBS and 0.05 mM 2-ME)
• 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 IL-4, clone 11B11 (Ultra-LEAF™ format, Cat. No. 504122)
• Anti-mouse IFN-γ, clone XMG1.2 (Ultra-LEAF™ format, Cat. No. 505834)
• Recombinant mouse IL-6 (carrier-free) (Cat. No. 575704)
• Recombinant mouse IL-23 (carrier-free) (Cat. No. 589002)
• Recombinant human TGF-β1 (carrier-free) (Cat. No. 781802)
• Brefeldin A (Cat. No. 420601)
• Monensin Solution (Cat. No. 420701)
• PMA (Phorbol 12-myristate 13-acetate) (Cat. No. P8139 from Sigma)
• Ionomycin (Cat. No. I0634 from Sigma)

References:

1. Cua DJ. et al. 2010. Nat Rev Immunol. 10: 479.
2. Pappu R. et al. 2010. J Clin Immunol. 30:185.
3. Kennedy, J. et al., 1996. J. Interferon Cytokine Res. 16: 611.
4. Schubert, D. et al., 2004. J. Immunol. 172: 4503.
5. Infante-Duarte, C. et al., 2000. J. Immunol. 165: 6107.
6. Harrington, L. E. et al., 2005. Nature Immunol. 6:1123.
7. Nekrasova, T. et al., 2005. J. Immunol. 175: 2734.
8. Yen, D. et al., 2006. J. Clin. Invest. 116: 1310.
9. Ehirchiou, D. et al., 2007. J. Exp. Med. 204:1519.
10. Kang, S. G. et al., 2007. J. Immunol. 179:3724.
11. Harrington, L. E. et al., 2005. Nature Immunol. 6:1123.

Data:

Naïve BALB/c mice 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), 50 ng/ml mouse IL-6 (Cat. No. 575704, 5 ng/ml mouse IL-23 (Cat. No. 589002), 1 ng/ml TGF-β1 (Cat. No. 781802), 10 µg/ml anti-mouse IL-4 (clone 11B11. Cat. No. 504121), 10 µg/ml anti-mouse IFN-γ (clone XMG1.2, Cat. No. 505833) for 4 days. On day 3, 5 ml of fresh media was added along with same the concentration of antibodies/cytokines as used on day 0. On day 4, the cells were re-stimulated with 500 ng/ml PMA/ionomycin in the presence of BFA (if detecting IL-21, monensin is recommended instead) for 6 hours. Then the cells were harvested and surface stained with CD4 FITC (clone GK1.5, Cat. No. 100405), intracellular stained with IL-17 PE (clone TC11-18H10.1, Cat. No. 506904) along with IFN-γ APC (clone XMG1.2 , Cat. No. 505809), IL-4 APC (clone 11B11, Cat. No. 504106), IL-9 APC (clone RM9A4, Cat. No. 514105) , IL-21 Alexa Fluor® 647, or IL-22 Alexa Fluor® 647 (clone Poly5164, Cat. No. 516406) after fixation and permeabilization.

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.