Th17 cell "deforming" guidelines: interesting tips for differentiation experiments that novices can also understand

I. Introduction of Th17 cells

Th17 cells are an important subset of CD4+ helper T cells, named for their predominant secretion of IL-17, regulated by the transcription factor RORγt and require TGF-β, IL-6 to initiate differentiation and IL-23 to maintain function. It can resist extracellular bacteria and fungi by recruiting neutrophils, especially protect mucosal tissues, which is the key to antimicrobial immunity; however, excessive activation can trigger excessive inflammation, which is closely related to autoimmune diseases such as rheumatoid arthritis and psoriasis, as well as inflammatory bowel disease, and it is an important target for the treatment of related diseases.

Figure 1. Th17 Cell Differentiation Diagram[1]

Figure 1. Th17 Cell Differentiation Diagram[1]

 

II. Guidelines for "Deformation" of Th17 Cells

Today our task is to successfully "transform" a naive "recruit" (naive CD4+ T cells) into a unique "inflammatory warrior"—the Th17 cells. Don 't worry, this guide will take you through this classic immunology experiment in the most interesting way.

1. Task Objectives and Equipment List

Terminal task: successful induction of naive CD4+ T cells into Th17 cells in culture dishes with specific combinations of "signaling factors" and verification of their identity by scientific methods.

Table1 Core Equipment List

Equipment type

Name of equipment

Annotation

Seed cell

Highly purified naive CD4 + T cells

Sorted from spleen or peripheral blood with > 90% purity is critical for success.

Initiation signals

TGF-β and IL-6

Synergistically initiate the Th17 differentiation program and upregulate the key transcription factor RORγt.

Enhancing signal

IL-23

Promotes expansion, functional stabilization, and survival of Th17 cells.

Shielded signals

Anti-IL-4 and anti-IFN-γ antibodies

Neutralizing murmur signals that mislead cells to differentiate into Th2 or Th1.

Activation signals

Anti-CD3/CD28 antibodies

Pre-coated on the bottom of the plate to provide antigen stimulation necessary for T cell activation.

Logistics replenishment

Complete RPMI 1640 medium

Provide all nutrients and suitable environment required for cell growth.

2. Three-step "deformation" method

Figure 2. Flow Chart of Th17 Differentiation Assay

Figure 2. Flow Chart of Th17 Differentiation Assay

Step 1: Activated ("Enlistment Mobilization")

Highly purified naive CD4 + T cells were obtained by magnetic bead sorting (MACS) or flow sorting (FACS) from mouse spleen or human peripheral blood. Sorted "recruits" (naive CD4 + T cells) were placed into an already established "training arena" (pre-coated plates with anti-CD3/anti-CD28 antibodies) to allow the cells to be effectively activated and enter a state of readiness.

Step 2: Induced differentiation ("specialty training")

Join our matching Specialty Training Package (cytokines and antibodies) right away while cells are activating.

Human System: TGF-β (3-5 ng/mL) + IL-6 (30-50 ng/mL) + IL-23 (20-30 ng/mL) + neutralizing antibody (1-10 μg/mL each)

Mouse System: TGF-β (1-3 ng/mL) + IL-6 (20-30 ng/mL) + IL-23 (10-20 ng/mL) + neutralizing antibody (1-10 μg/mL each)

Step 3: Culture metamorphosis ("waiting to grow")

The cells were incubated at 37℃ in an incubator containing 5% CO2 for 3-5 days. Fresh medium (containing cytokines) may be changed in half every 1-2 days to maintain signal strength and provide nutrients.

3. Acceptance Results: How to identify your "Th17 warriors"?

After training is completed, it is necessary to check how many cells have been successfully "transferred".

Identification methods

Test target

Interpretation of Results

Advantages

Flow cytometry (gold standard)

Intracellular

IL-17A protein

Percentage of IL-17A positive cells in cell population

Direct, quantitative, can be analyzed at single cell level

intranuclear Transcription Factor RORγt

Percentage of cells expressing RORγt

Detect "identity commander", earlier and more essential

ELISA

IL-17A in Culture Supernatants

Concentration of IL-17A in supernatant

 

Functional validation, relatively simple operation, batch testing

 

�� Novice pit avoidance tips

Purity first: Impurity of naive T cells is the primary culprit in experimental failure, and it is important to ensure sorting purity.

Cytokine activity: Ensure the activity and concentration of cytokines, which are the direct driving force for successful induction.

Do not forget "Scavengers": Anti-IL-4/anti-IFN-γ antibodies must be added, otherwise cells will be very susceptible to differentiation into other types.

Control group: It is necessary to set a negative control group without inducing factor (activated only) for a convincing comparison!

 

Now, you have mastered a full training program from "recruits" to "warriors". Get ready to start your cell "transformation" experiment with Yeasen’s HiActi cytokines!

Ⅲ、Yeasen HiActi Cytokines

Yeasen has developed a series of HiActi® cytokines specifically designed for cell culture. Strict quality control and validation of cellular functions have ensured that the products have high activity, high purity, high stability, and low endotoxin levels. The key cytokines that enable Th17 differentiation are IL-6, IL-21 (which maintains Th17 cell differentiation in the form of autocrine) and TGF-β, while IL-23 plays an important role in the maintenance and proliferation of Th17. Yeasen’s cytokines can precisely assist the differentiation of Th17 cells and help you to obtain ideal experimental results.

Product data

  • Bioactivity of human IL-6
Figure 3. The ED50 as determined by a cell proliferation assay using human TF-1 cells is 0.1-0.5 ng/mL.

Figure 3. The ED50 as determined by a cell proliferation assay using human TF-1 cells is 0.1-0.5 ng/mL.

  • Bioactivity of Human/Mouse/Rat TGF-beta 1
Figure 4. Measured by the (CAGA)12—luciferase reporter assay. The EC50 for this effect is 1.092 ng/mL.

Figure 4. Measured by the (CAGA)12—luciferase reporter assay. The EC50 for this effect is 1.092 ng/mL.

  • Bioactivity of Human IL-23
Figure 5. Measured by its ability to induce IL-17 secretion by mouse splenocytes. The EC50 for this effect is 0.25 pg/mL.

Figure 5. Measured by its ability to induce IL-17 secretion by mouse splenocytes. The EC50 for this effect is 0.25 pg/mL.

Ordering Infomation

Product name

Item No.

Strength

Recombinant Human IL-6 Protein

90107ES

5 μg/20 μg/50 μg/100 μg/1 mg

Recombinant Mouse IL-6 Protein

90146ES

2 μg/10 μg/50 μg/100 μg/500 μg

Human/Mouse/Rat Recombinant TGF-beta 1/TGF-β1 Protein

91701ES

2 μg/10 μg/100 μg

Recombinant Human TGF-β2 Protein (CHO)

91709ES

10 μg/50 μg/1 mg

Recombinant Human TGF-beta 3 Protein

91705ES

10 μg/50 μg/100 μg

Recombinant Human IL-23 Protein (liquid)

90225ES

10 μg/100 μg/1 mg

Recombinant Mouse IL-23 Protein

90226ES

10 μg/50 μg/100 μg/1 mg

 

References

1. McGeachy MJ, Cua DJ. Th17 Cell Differentiation: The Long and Winding Road. Immunity 2008; 28(4):445-453.

 

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