Treg

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.